Novel matrix metalloproteinases

ABSTRACT

The present invention provides genes encoding novel matrix metalloproteinases termed MMP; constructs and recombinant host cells incorporating the genes; the MMP polypeptides encoded by the genes; antibodies to the MMP polypeptides; and methods of making and using all of the foregoing.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority of application Ser. No.60/206,119, filed May 22, 2000 which is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the fields of geneticsand cellular and molecular biology. More particularly, the inventionrelates to novel matrix metalloproteinases.

BACKGROUND OF THE INVENTION

[0003] Matrix metalloproteinases (MMPs; matrixins) comprise a family ofstructurally related Zn-containing proteases that degrade allmacromolecules present in the extracellular matrix (ECM). Each of theknown MMPs can be divided up into a variable number of well-conserveddomains. All contain a pro-peptide which is involved in suppressing theactivity of the pro-enzyme form of the molecule, and a HEXXH sequencemotif that has been shown by X-ray crystallography to form part of themetal-binding site (Nagase et al. (1999) J. Biol. Chem., 274, 31,21491-21494). In addition, fibronectin-, hemopexin-, or vitronectin-likedomains and/or a membrane “anchor” domain may also be present.

[0004] Today, the MM family includes more than 15 members (Table 1).TABLE 1 Characteristics of known human MMPs MMP Type Substrate MMP-1Collagenase collagen I, II, III, VII, X gelatins MMP-2 Gelatinase Bcollagens IV, V, VII, XI, fibronectin, elastin MMP-3 Stromelysin 1proteoglycans, gelatins, fibronectin, collagens II, IV, IX MMP-7Matrilysin Proteoglycans, gelatins, collagen TV, elastin MMP-8Neutrophil collagens I, II, III collagenase MMP-9 Gelatinase B gelatins,collagen IV, V, proteoglycan, elastin MMP-10 Stromelysin 2 proteoglycan,fibronectin, laminin MMP-12 Macrophage proteoglycans, elastase elastaseMMP-13 Collagenase 3 collagens I, II, III, IV

[0005] MMPs are believed to play a critical role in many physiologicaland pathological processes. The breakdown of ECM by MMPs is essentialfor processes including embryonic development, morphogenesis,reproduction, and tissue repair and remodeling. Other physiologicalprocesses which involve MMPs include tumor growth, tumor invasion,Sjögren's syndrome, periodontal diseases, arthritis, cardiomyopathy,renal failure, atherosclerosis, insulin resistance, adipogenesis,retinal neovascularization, wound healing, and neurodegenerativediseases including, for example, Alzheimer's disease, multiplesclerosis, Parkinson's disease, and motoneuron disease. Identificationof the functions of additional genes of the MMP family would beinvaluable to those of skill in the art seeking to understand thegenetic basis for these processes, as well as identifying compounds thatmodulate the activity of such genes useful in methods for treating thepathologies.

[0006] Tissue inhibitors of metalloproteinases (TIMPs) are a group ofclosely related secreted proteins that limit MMP activity. To date, fourTIMPs have been characterized; TIMP1, TIMP2, TIMP3, and TIMP4,respectively (Gomez et al. (1997) Eur. J. Cell. Biol., 74, 111-122).Several investigators have studied effects of MMP inhibition by usingcells over-expressing TIMPs. The balance between MMPs and TIMPs seems toplay an important role in matrix turnover in several organ systems.

[0007] The past few years have witnessed several advances in theunderstanding of the pathophysiology of coronary atherosclerosis. Theearliest atherosclerotic lesion, named the fatty streak, represents adynamic balance of the entry and exit of lipoprotein as well as thedevelopment of extracellular matrix. A decrease in lipoprotein entrywill probably result in a predominance of lipoprotein exit and finalscarring. However, an increase of lipoprotein entry can predominate overthe efflux and scarring, resulting in vulnerable lipid-rich plaques thatare prone to disruption (Falk et al., (1995), Circulation, 92:657-671;Fuster et al., (1999), Lancet, 353:SII: 5-9).

[0008] It is evident from many studies that MMPs, as a family, areimportant regulators of atherosclerotic plaque growth (Newby et al.,(1994), Basic Res. Cardiol. 89 [Suppl. 1] 59-70). However, the roles ofthe individual MMPs are so far largely unknown. Several MMPs areexpressed in the diseased blood vessel, i.e. in smooth muscle cells andin macrophages. MMPs likely regulate both the degradation ofextracellular matrix and influence the proliferation rate of smoothmuscle cells. Several inflammatory cytokines and growth factors increasethe expression of MMPs in cell cultures, e.g. interleukin-1,platelet-derived growth factor (PDGF) and tumor necrosis factor-α(TNF-α).

[0009] It has been demonstrated in several animal models that inhibitionof MMPs (type 1 and 2 among others) decreases smooth muscleproliferation in response to vascular damage. Moreover, MMPs seem toenhance smooth muscle cell migration. These two physiological processesare hallmarks of the neointimal thickening that characterizesatherosclerosis. Accordingly, MMP inhibitors may delay or preventspontaneous atherogenesis as well as restenosis. MMPs and/or TIMPs maybe especially useful for patients at risk for atherosclerosis,dyslipidemia, end-stage renal failure, or patients who have undergonePercutaneous Transluminal Coronary Angioplasty Procedure (PTCA).

[0010] A large number of studies support a role of MMPs in intima mediafunction. For example, over-expression of TIMP2 inhibits vascular smoothmuscle cell proliferation and chemotaxis in vitro (Baker et al., (1998),J. Clin. Invest., 101:1478-1487; Cheng et al., (1998), Circulation,98:2195-2201). In addition, it has been shown that MMPs are linked tothe proliferation and outgrowth of vascular smooth muscle cells fromexplants of rabbit aorta in vitro. The proliferation and outgrowth ofvascular smooth muscle cells from rabbit aorta was blocked byexperimental inhibitors (Ro 31-4724 and Ro 31-7467) (Newby et al.,(1994). Batimastat (BB94), a synthetic MMP inhibitor, can reduce smoothmuscle cell proliferation in vitro as well as inhibit neointimalformation after balloon injury to the rat carotid artery (Zempo et al.,(1996), Artherioscler. Thromb. Vasc. Biol., 16:28-33). Localoverexpression of TIMP1 has been shown to inhibit intimal hyperplasia inrats (Forough et al., (1996), Circ. Res., 79:812-820). After in vitroincubation with MMP-3, -7, or -12, the ability of HDL(3) to induce thehigh affinity component of cholesterol efflux from the macrophage foamcells was strongly reduced (Lindstedt et al., (1999), J. Biol. Chem.,274:22627-22634).

[0011] Angiogenesis, also known as neovascularization or new vesselgrowth, is part of the normal wound healing machinery and can occur as areaction to tissue hypoxia. Various tumors are also known to triggerangiogenesis, leading to tumor growth. In normal adult tissue, there isa balance between angiogenic and anti-angiogenic factors and, as aresult, few new vessels are formed. However, if the balance betweenangiogenic and anti-angiogenic factors is disturbed, a complex cascadeof events can be triggered that eventually leads to the formation of newblood vessels.

[0012] Diabetic retinopathy is the leading cause of blindness for themajority of Americans. Microvascular damage from diabetes leads tomicroaneurysms, hemorrhage, exudates, and cotton-wool spots. Furtherprogression of disease leads to neovascularization. Growth of new bloodvessels can cause severe hemorrhage, scarring, and permanent visual loss(for a review, see Frank et al, (1996), South. Med. J., 89:463-470;Jampol & Goldbaum, (1980), Surv. Ophthalmol. 25:1-14). Variousrandomized, prospective studies have clearly shown benefit from lasertherapy at specific stages of progression of retinopathy.

[0013] AMD with rapid progression (wet AMD) is another common cause ofblindness in the developed world. Presently the underlying etiology ofAMD is unknown but a slow deterioration of the retinal pigmentepithelium, leading to the death of macular photoreceptors, is believedto be an important factor. The wet form of AMD often leads to a completeloss of central vision within a few years. AMD usually debuts in the dryform and may subsequently change into the wet form. AMD with rapidprogression is characterized by choroidal new vessel formation (CNV).The new vessels tend to leak and may rupture. The resulting macularedema, bleeding, fibrinous deposits, and scar formation are reasons forthe rapid deterioration of vision in this form of AMD.

[0014] Sprouting is a key step in CNV formation. If sprouting can beinhibited, no new leaky vessels will form. MMPs are essential to createspace for the new sprouts. Because this step is downstream in theangiogenesis process, an MMP inhibitor can work to limit sprouting evenif the earlier events are slightly different from those described above.The localization of MMP-2 and MMP-9 to the areas of new vessel formationand to the enveloping Bruch's-like membrane, respectively, suggests thatMMP-2 and MMP-9 may be cooperatively involved in the progressive growthof choroidal neovascular membranes (Steen et al. (1998), Invest.Ophthalmol. Vis. Sci., 39:2194-2200). In normal individuals MMP-9activity is not detected in the eye; however, it has been demonstratedthat MMP-9 activity is detected in more than 80% of patients with“active” proliferative retinopathy (Kosano et al., (1999), Life Sci.,64:2307-2315).

[0015] MMP inhibitors present an attractive opening for prophylacticpharmacotherapy of ocular blood vessel proliferation in diabetes andAMD. Moreover, it may be possible to combine MMP inhibitors withphotodynamic therapy. It is possible that inhibitors of MMPs couldprevent recurrence of CNV and, thus, improve long-term efficacy.Patients are likely to accept certain side effects in order to preservetheir vision, as most are aware that the disease will rapidly lead toblindness. Topical treatment is advantageous from a pharmacovigilancepoint-of-view.

[0016] MMPs are also involved in the bioacticvation of cytokines,including tumor necrosis factor-alpha (TNF-α). Evidence suggests thatTNF-α is a key mediator of insulin resistance in adipocytes and skeletalmuscle. Inhibition of MMPs may decrease the formation rate of TNF-α and,accordingly, be of therapeutic significance in type-II diabetes. MMPsand/or TIMPS may be useful for patients with Type II diabetes or forobese patients with insulin resistance.

[0017] It has been suggested that TNF-α is an inducer of insulinresistance in type II diabetes. TNF-α is synthesized as a membrane-boundprecursor that is proteolytically processed to an active form by amatrix metalloproteinase (MMP)-like enzyme. It has been shown thatsubcutaneous administration of KB-R7785 (a non-specific MMP inhibitor)to KKAy mice, which show insulin resistance and hyperglycemia for 4weeks, resulted in a significant decrease in plasma glucose levels after3 weeks of administration. In the same study it was also demonstratedthat administration of pioglitazone significantly decreased plasmaglucose levels. Interestingly, KB-R7785, but not pioglitazone, alsosignificantly decreased plasma insulin levels in the animals. It hasalso been shown that the lipopolysaccharide-induction of TNF-α in plasmacan be inhibited in KKAy mice by KB-R7785. These results suggest thatMNMP inhibitors may exert an anti-diabetic effect by amelioratinginsulin sensitivity through the inhibition of TNF-α production.

[0018] Nephropathy in patients with type I and II diabetes mellitus is arapidly increasing problem worldwide. Diabetic patients account fornearly half of all patients on hemodialysis. Microalbuminuria isdiagnosed when the urinary albumin excretion rate is greater than 20 butless than 200 micrograms/min and the prevalence of microalbuminuriaamong diabetic patients is 15-20% (Deckert et al., (1992), DiabetesCare, 15:1181-1191).

[0019] MMP inhibitors (non-selective) have been found to decrease theproliferation rate of cultured rat mesangial cells without affectingcell viability. Therefore, MMP inhibitors may offer a new therapeuticapproach for treatment of mesangial cell-derived forms ofglomerulonephritis and prevent basal membrane thickening in diabetes.MMPs and/or TIMPS may be useful for diabetic patients with early signsof glomerulopathy, or for patients with microalbuminuria.

[0020] Progressive expansion of the mesangial matrix, and thickening ofthe glomerular and tubular basement membranes are hallmarks of human andexperimental diabetic nephropathy (Philips et al. (1999), Kidney BloodPress. Res. 22:81-97; Young et al., (1995) Kidney Int., 47:935-944).These lesions eventually lead to glomerular fibrosis, a centralpathological feature in many human acute and chronic kidney diseases,which progressively destroys the renal filtration unit, and may finallycause renal failure. It has been demonstrated that mesangial matrixexpansion is strongly related to the clinical manifestation of diabeticnephropathy. Diabetic nephropathy is effected both directly andindirectly by the alteration of cytokine generation. Data from studieson several animal species suggest that proliferation of mesangial cellsis an important feature of diabetic glomerulopathy. Harendza et al.,(Nephrol. Dial. Transplant 12:2537-2541, (1997)) have demonstrated thatthe expression of MMP2 is enhanced in experimental proliferativeglomerulopathy in the rat. Inhibition of MMP2 by Ro 31-9790 inhibitedthe proliferation rate of cultured rat mesangial cells in aconcentration-dependent and at least partially reversible manner withoutaffecting cell viability (Steinmann-Niggli K, et al., (1997), J. Am.Soc. Nephrol. 8:395-405). Moreover, Ebihara et al., ((1998) Am. J.Kidney Dis. 32:544-550) have reported that increased MMP9 concentrationsin plasma preceded the occurrence of microalbuminuria in diabeticpatients.

[0021] Thus, a need exists for new members of the MMP family ofproteases.

SUMMARY OF THE INVENTION

[0022] The present invention addresses the need identified above byproviding DNA sequences of genes encoding heretofore unknown members ofthe MMP family of proteases, bearing sequence homology and functionalhomology to MMPs; constructs and recombinant host cells incorporatingthe genes; the MMP polypeptides encoded by the genes; antibodies to thepolypeptides; kits employing the polynucleotides and polypeptides, andmethods of making and using all of the foregoing. Exemplary diseases andconditions amenable to treatment based on the present invention include,but are not limited to metabolic diseases and disorders (e.g., type 2diabetes, obesity, cardiovascular, dyslipidemias, adipogenesis,retinopathies, neuropathies, nephropathies etc.), proliferative diseasesand cancers (e.g., different cancers such as breast, colon, lung, etc.,tumor growth, tumor invasion, and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc.), hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.), CNS disorders (e.g., degenerative disorders such as Parkinson's,Alzheimer's, etc.), inflammatory conditions (e.g., Chron's disease,arthritis), diseases related to cell differentiation and homeostasis,cardiomyopathy, atherosclerosis, thromboembolic diseases, Sjögren'ssyndrome, renal failure, periodontal diseases, retinalneovascularization, wound healing, and neurodegenerative diseasesincluding, for example, Alzheimer's disease, multiple sclerosis,Parkinson's disease, and motoneuron disease, among others.

[0023] In one embodiment, the invention provides purified and isolatedMMP polypeptides comprising the amino acid sequence set forth in SEQ IDNOS: 4-6, or a fragment thereof comprising an epitope specific to theMMP polypeptide. Preferred embodiments comprise purified and isolatedpolypeptides comprising the complete amino acid sequences set forth inany of SEQ ID NOS: 4-6, found in Table 5 below. These amino acidsequences were deduced from the polynucleotide sequences encodingMMP-(SEQ ID NOS: 1-3 found in Table 5 below).

[0024] In another preferred embodiment, the invention provides apurified and isolated polypeptide comprising at least one conserved MMPdomain.

[0025] In another embodiment, the invention provides purified andisolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA,or combinations thereof, whether single- or double-stranded) thatcomprise a nucleotide sequence encoding the amino acid sequence of thepolypeptides of the invention. Such polynucleotides are useful forrecombinantly expressing the protease and also for detecting expressionof the protease in cells (e.g., using Northern hybridization and in situhybridization assays. Such polynucleotides also are useful in the designof antisense and other molecules for the suppression of the expressionof MMPs in a cultured cell, a tissue, or an animal; for therapeuticpurposes; or to provide a model for diseases or conditions characterizedby aberrant MMP expression. Specifically excluded from the definition ofpolynucleotides of the invention are entire isolated, non-recombinantnative chromosomes of host cells. Preferred polynucleotides have thesequences set forth in SEQ ID NOS: 1-3, which correspond to a naturallyoccurring MMP-sequences.

[0026] The invention also provides a purified and isolatedpolynucleotide comprising a nucleotide sequence that encodes a mammaliangene product, wherein the polynucleotide hybridizes to a polynucleotidehaving the sequences set forth in SEQ ID NOS: 1-3 or the non-codingstrand complementary thereto, under the following hybridizationconditions:

[0027] (a) hybridization for 16 hours at 42° C. in a hybridizationsolution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextransulfate; and

[0028] (b) washing 2 times for 30 minutes each at 60° C. in a washsolution comprising 0.1% SSC, 1% SDS. Polynucleotides that encode ahuman allelic variant are highly preferred.

[0029] In a related embodiment, the invention provides vectorscomprising a polynucleotide of the invention. Such vectors are useful,e.g., for amplifying the polynucleotides in host cells to create usefulquantities thereof. In preferred embodiments, the vector is anexpression vector wherein the polynucleotide of the invention isoperatively linked to a polynucleotide comprising an expression controlsequence. Such vectors are useful for recombinant production ofpolypeptides of the invention.

[0030] In another related embodiment, the invention provides host cellsthat are transformed or transfected (stably or transiently) withpolynucleotides of the invention or vectors of the invention. Such hostcells are useful for amplifying the polynucleotides and also forexpressing the MMP polypeptides or fragments thereof encoded by thepolynucleotides.

[0031] In still another related embodiment, the invention provides amethod for producing MMP polypeptides (or fragments thereof) comprisingthe steps of growing a host cell of the invention in a nutrient mediumand isolating the polypeptide or variant thereof from the cell or themedium.

[0032] In still another embodiment, the invention provides an antibodythat is specific for the MMP of the invention. Antibody specificity isdescribed in greater detail below. However, it should be emphasized thatantibodies that can be generated from polypeptides that have previouslybeen described in the literature and that are capable of fortuitouslycross- reacting with MMP (e.g., due to the fortuitous existence of asimilar epitope in both polypeptides) are considered “cross-reactive”antibodies. Such cross-reactive antibodies are not antibodies that are“specific” for MMP.

[0033] In one preferred variation, the invention provides monoclonalantibodies. Hybridomas that produce such antibodies also are intended asaspects of the invention. In yet another variation, the inventionprovides a humanized antibody. Humanized antibodies are useful for invivo therapeutic indications.

[0034] In another variation, the invention provides a cell-freecomposition comprising polyclonal antibodies, wherein at least one ofthe antibodies is an antibody of the invention specific for MMP.Antisera isolated from an animal is an exemplary composition, as is acomposition comprising an antibody fraction of an antisera that has beenresuspended in water or in another diluent, excipient, or carrier.

[0035] In still another related embodiment, the invention provides ananti-idiotypic antibody specific for an antibody that is specific forMMP.

[0036] In still another embodiment, the invention provides a polypeptidecomprising a fragment of an MMP-specific antibody, wherein the fragmentand the polypeptide bind to the MMP. By way of non-limiting example, theinvention provides polypeptides that are single chain antibodies andCDR-grafted antibodies.

[0037] Also within the scope of the invention are compositionscomprising polypeptides, polynucleotides, or antibodies of the inventionthat have been formulated with, e.g., a pharmaceutically acceptablecarrier.

[0038] The invention also provides methods of using antibodies of theinvention. For example, the invention provides a method for modulatingsubstrate binding of a MMP comprising the step of contacting the MMPwith an antibody specific for the MMP, under conditions wherein theantibody binds the MMP.

[0039] MMPs may be expressed in various tissues, and an expressionprofile of such tissues provides additional uses for the invention. Forexample, if MMP is expressed in the brain, it would provide anindication that aberrant MMP activity may correlate with one or moreneurological disorders. The invention thus also provides a method fortreating a neurological disorder comprising the step of administering toa mammal in need of such treatment an amount of an antibody-likepolypeptide of the invention that is sufficient to modulate substratebinding to a MMP in neurons of the mammal. MMP may also be expressed inother tissues, including but not limited to pancreas (and particularlypancreatic islet tissue), pituitary, skeletal muscle, adipose tissue,liver, and thyroid.

[0040] The invention also provides assays to identify compounds thatbind a MMP. One such assay comprises the steps of: (a) contacting acomposition comprising a MMP with a compound suspected of binding MMP;and (b) measuring binding between the compound and MMP. In onevariation, the composition comprises a cell expressing MMP.

[0041] The invention also provides a method for identifying a modulatorof binding between a MMP and a MMP binding partner, comprising the stepsof: (a) contacting a MMP binding partner and a composition comprising aMMP in the presence and in the absence of a putative modulator compound;(b) detecting binding between the binding partner and the MMP; and (c)identifying a putative modulator compound or a modulator compound inview of decreased or increased binding between the binding partner andthe MMP in the presence of the putative modulator, as compared tobinding in the absence of the putative modulator.

[0042] MMP binding partners that stimulate MMP activity are useful asactivators in disease states or conditions characterized by insufficientMMP activity. MMP binding partners that block MMP-mediated proteolysisare useful as MMP repressors to treat disease states or conditionscharacterized by excessive MMP-mediated proteolysis. In addition MMPproteolysis modulators in general, as well as MMP polynucleotides andpolypeptides, are useful in diagnostic assays for such diseases orconditions.

[0043] In another aspect, the invention provides methods for treating adisease or abnormal condition by administering to a patient in need ofsuch treatment a substance that modulates the activity or expression ofMMP. Preferably, the disease is selected from the group consisting oftumor growth, tumor invasion, Sjögren's syndrome, periodontal diseases,arthritis, cardiomyopathy, renal failure, atherosclerosis, insulinresistance, adipogenesis, retinal neovascularization, wound healing, andneurodegenerative diseases including, for example, Alzheimer's disease,multiple sclerosis, Parkinson's disease, and motoneuron disease.

[0044] In another aspect, the invention features methods for detectionof a polypeptide in a sample as a diagnostic tool for diseases ordisorders, wherein the method comprises the steps of: (a) contacting thesample with a nucleic acid probe which hybridizes under hybridizationassay conditions to a nucleic acid target region of a MMP polypeptide,said probe comprising the nucleic acid sequence encoding thepolypeptide, fragments thereof, and the complements of the sequences andfragments; and (b) detecting the presence or amount of the probe:targetregion hybrid as an indication of the disease.

[0045] The diseases for which detection of genes in a sample could bediagnostic include diseases in which nucleic acid (DNA and/or RNA) isamplified in comparison to normal cells. The diseases that could bediagnosed by detection of nucleic acid in a sample preferably includecancers. The test samples suitable for nucleic acid probing methods ofthe present invention include, for example, cells or nucleic acidextracts of cells, or biological fluids. The samples used in theabove-described methods will vary based on the assay format, thedetection method and the nature of the tissues, cells or extracts to beassayed. Methods for preparing nucleic acid extracts of cells are wellknown in the art and can be readily adapted in order to obtain a samplethat is compatible with the method utilized.

[0046] Additional features and variations of the invention will beapparent to those skilled in the art from the entirety of thisapplication, including the detailed description, and all such featuresare intended as aspects of the invention. Likewise, features of theinvention described herein can be re-combined into additionalembodiments that also are intended as aspects of the invention,irrespective of whether the combination of features is specificallymentioned above as an aspect or embodiment of the invention. Also, onlysuch limitations that are described herein as critical to the inventionshould be viewed as such; variations of the invention lackinglimitations that have not been described herein as critical are intendedas aspects of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0047] The present invention provides purified and isolatedpolynucleotides (e.g., DNA sequences and RNA transcripts, both sense andcomplementary antisense strands, both single- and double-stranded,including splice variants thereof) encoding a matrix metalloproteinasereferred to herein as MMP. DNA polynucleotides of the invention includegenomic DNA, cDNA, and DNA that has been chemically synthesized in wholeor in part.

[0048] Definitions

[0049] Various definitions are made throughout this document. Most wordshave the meaning that would be attributed to those words by one skilledin the art. Words specifically defined either below or elsewhere in thisdocument have the meaning provided in the context of the presentinvention as a whole and as are typically understood by those skilled inthe art.

[0050] “Synthesized” as used herein and understood in the art, refers topolynucleotides produced by purely chemical, as opposed to enzymatic,methods. “Wholly” synthesized DNA sequences are therefore producedentirely by chemical means, and “partially” synthesized DNAs embracethose wherein only portions of the resulting DNA were produced bychemical means.

[0051] By the term “region” is meant a physically contiguous portion ofthe primary structure of a biomolecule. In the case of proteins, aregion is defined by a contiguous portion of the amino acid sequence ofthat protein.

[0052] The term “domain” is herein defined as referring to a structuralpart of a biomolecule that contributes to a known or suspected functionof the biomolecule. Domains may be co-extensive with regions or portionsthereof; domains may also incorporate a portion of a biomolecule that isdistinct from a particular region, in addition to all or part of thatregion.

[0053] As used herein, the terms “ligand” and “binding partner” are usedinterchangeably and refer to compounds that bind to proteases such asMMP or portions of such proteases.

[0054] Unless indicated otherwise, as used herein the abbreviation inlower case (mmp) refers to a gene, cDNA, RNA or nucleic acid sequencewhile the upper case version (MMP) refers to a protein, polypeptide,peptide, oligopeptide, or amino acid sequence.

[0055] As used herein, the term “activity” refers to a variety ofmeasurable indicia suggesting or revealing binding, either direct orindirect; affecting a response, i.e. having a measurable affect inresponse to some exposure or stimulus, including, for example, theaffinity of a compound for directly binding a polypeptide orpolynucleotide of the invention, or, for example, measurement of amountsof upstream or downstream proteins or other similar functions after somestimulus or event.

[0056] As used herein, the term “antibody” is meant to refer tocomplete, intact antibodies, and Fab, Fab′, F(ab)2, and other fragmentsthereof. Complete, intact antibodies include monoclonal antibodies suchas murine monoclonal antibodies, chimeric antibodies and humanizedantibodies.

[0057] As used herein, the term “binding” means the physical or chemicalinteraction between two proteins or compounds or associated proteins orcompounds or combinations thereof. Binding includes ionic, non-ionic,Hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Thephysical interaction, the binding, can be either direct or indirect,indirect being through or due to the effects of another protein orcompound. Direct binding refers to interactions that do not take placethrough or due to the effect of another protein or compound but insteadare without other substantial chemical intermediates. Binding may bedetected in many different manners. As a non-limiting example, thephysical binding interaction between a MMP of the invention and acompound can be detected using a labeled compound. Alternatively,functional evidence of binding can be detected using, for example, acell transfected with and expressing an MMP of the invention. Binding ofthe transfected cell to a ligand of the MMP that was transfected intothe cell provides functional evidence of binding. Other methods ofdetecting binding are well known to those of skill in the art.

[0058] As used herein, the term “compound” means any identifiablechemical or molecule, including, but not limited to, small molecule,peptide, protein, sugar, nucleotide, or nucleic acid, and such compoundcan be natural or synthetic.

[0059] As used herein, the term “complementary” refers to Watson-Crickbasepairing between nucleotide units of a nucleic acid molecule.

[0060] As used herein, the term “contacting” means bringing together,either directly or indirectly, a compound into physical proximity to apolypeptide or polynucleotide of the invention. The polypeptide orpolynucleotide can be in any number of buffers, salts, solutions etc.Contacting includes, for example, placing the compound into a beaker,microtiter plate, cell culture flask, or a microarray, such as a genechip, or the like, which contains the nucleic acid molecule, orpolypeptide encoding the MMP or fragment thereof.

[0061] As used herein, the phrase “homologous nucleotide sequence,” or“homologous amino acid sequence,” or variations thereof, refers tosequences characterized by a homology, at the nucleotide level or aminoacid level, of at least the specified percentage. Homologous nucleotidesequences include those sequences coding for isoforms of proteins. Suchisoforms can be expressed in different tissues of the same organism as aresult of, for example, alternative splicing of RNA. Alternatively,isoforms can be encoded by different genes. Homologous nucleotidesequences include nucleotide sequences encoding for a protein of aspecies other than humans, including, but not limited to, mammals.Homologous nucleotide sequences also include, but are not limited to,naturally occurring allelic variations and mutations of the nucleotidesequences set forth herein. A homologous nucleotide sequence does not,however, include the nucleotide sequence encoding other known MMPs.Homologous amino acid sequences include those amino acid sequences whichcontain conservative amino acid substitutions and which polypeptideshave the same binding and/or activity. A homologous amino acid sequencedoes not, however, include the amino acid sequence encoding other knownMMPs. Percent homology can be determined by, for example, the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, Madison Wis.), usingthe default settings, which uses the algorithm of Smith and Waterman(Adv. Appl. Math., 1981, 2, 482-489, which is incorporated herein byreference in its entirety).

[0062] As used herein, the term “isolated” nucleic acid molecule refersto a nucleic acid molecule (DNA or RNA) that has been removed from itsnative environment. Examples of isolated nucleic acid molecules include,but are not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules.

[0063] As used herein, the terms “modulates” or “modifies” means anincrease or decrease in the amount, quality, or effect of a particularactivity or protein.

[0064] As used herein, the term “oligonucleotide” refers to a series oflinked nucleotide residues which has a sufficient number of bases to beused in a polymerase chain reaction (PCR). This short sequence is basedon (or designed from) a genomic or cDNA sequence and is used to amplify,confirm, or reveal the presence of an identical, similar orcomplementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a DNA sequence having at leastabout 10 nucleotides and as many as about 50 nucleotides, preferablyabout 15 to 30 nucleotides. They are chemically synthesized and may beused as probes.

[0065] As used herein, the term “probe” refers to nucleic acid sequencesof variable length, preferably between at least about 10 and as many asabout 6,000 nucleotides, depending on use. They are used in thedetection of identical, similar, or complementary nucleic acidsequences. Longer length probes are usually obtained from a natural orrecombinant source, are highly specific and much slower to hybridizethan oligomers. They may be single- or double-stranded and carefullydesigned to have specificity in PCR, hybridization membrane-based, orELISA-like technologies.

[0066] The term “preventing” refers to decreasing the probability thatan organism contracts or develops an abnormal condition.

[0067] The term “treating” refers to having a therapeutic effect and atleast partially alleviating or abrogating an abnormal condition in theorganism.

[0068] The term “therapeutic effect” refers to the inhibition oractivation factors causing or contributing to the abnormal condition. Atherapeutic effect relieves to some extent one or more of the symptomsof the abnormal condition. In reference to the treatment of abnormalconditions, a therapeutic effect can refer to one or more of thefollowing: (a) an increase in the proliferation, growth, and/ordifferentiation of cells; (b) inhibition (i.e., slowing or stopping) ofcell death; (c) inhibition of degeneration; (d) relieving to some extentone or more of the symptoms associated with the abnormal condition; and(e) enhancing the function of the affected population of cells.Compounds demonstrating efficacy against abnormal conditions can beidentified as described herein.

[0069] The term “abnormal condition” refers to a function in the cellsor tissues of an organism that deviates from their normal functions inthat organism. An abnormal condition can relate to cell proliferation,cell differentiation, cell signaling, or cell survival. An abnormalcondition may also include obesity, diabetic complications such asretinal degeneration, and irregularities in glucose uptake andmetabolism, and fatty acid uptake and metabolism.

[0070] Abnormal cell proliferative conditions include cancers such asfibrotic and mesangial disorders, abnormal angiogenesis andvasculogenesis, wound healing, psoriasis, diabetes mellitus, andinflammation.

[0071] Abnormal differentiation conditions include, but are not limitedto, neurodegenerative disorders, slow wound healing rates, and slowtissue grafting healing rates. Abnormal cell signaling conditionsinclude, but are not limited to, psychiatric disorders involving excessneurotransmitter activity.

[0072] Abnormal cell survival conditions may also relate to conditionsin which programmed cell death (apoptosis) pathways are activated orabrogated. A number of protein kinases are associated with the apoptosispathways. Aberrations in the function of any one of the protein kinasescould lead to cell immortality or premature cell death.

[0073] The term “aberration,” in conjunction with the function of anMMP, refers to an MMP that is over- or under-expressed in an organism,mutated such that its catalytic activity is lower or higher thanwild-type protease activity, mutated such that it can no longer interactwith a natural binding partner, or is no longer modified by anotherprotease.

[0074] The term “administering” relates to a method of incorporating acompound into cells or tissues of an organism. The abnormal conditioncan be prevented or treated when the cells or tissues of the organismexist within the organism or outside of the organism. Cells existingoutside the organism can be maintained or grown in cell culture dishes.For cells harbored within the organism, many techniques exist in the artto administer compounds, including (but not limited to) oral,parenteral, dermal, injection, and aerosol applications. For cellsoutside of the organism, multiple techniques exist in the art toadminister the compounds, including (but not limited to) cellmicroinjection techniques, transformation techniques and carriertechniques.

[0075] The abnormal condition can also be prevented or treated byadministering a compound to a group of cells having an aberration in asignal transduction pathway to an organism. The effect of administeringa compound on organism function can then be monitored. The organism ispreferably a mouse, rat, rabbit, guinea pig or goat, more preferably amonkey or ape, and most preferably a human.

[0076] By “amplification” it is meant increased numbers of DNA or RNA ina cell compared with normal cells. “Amplification” as it refers to RNAcan be the detectable presence of RNA in cells, since in some normalcells there is no basal expression of RNA. In other normal cells, abasal level of expression exists, therefore in these cases amplificationis the detection of at least 1 to 2-fold, and preferably more, comparedto the basal level.

[0077] As used herein, the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a probe,primer, or oligonucleotide will hybridize to its target sequence, but tono other sequences. Stringent conditions are sequence-dependent and willbe different in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present in excess, at T_(m), 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g. 10 to 50 nucleotides) and at leastabout 60° C. for longer probes, primers or oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

[0078] The amino acid sequences are presented in the amino to carboxydirection, from left to right. The amino and carboxy groups are notpresented in the sequence. The nucleotide sequences are presented bysingle strand only, in the 5′ to 3′ direction, from left to right.Nucleotides and amino acids are represented in the manner recommended bythe IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids)by three letters code.

[0079] Polynucleotides

[0080] The present invention provides purified and isolatedpolynucleotides (e.g., DNA sequences and RNA transcripts, both sense andcomplementary antisense strands, both single- and double-stranded,including splice variants thereof) that encode unknown MMPs heretoforetermed novel MMPs, or MMPs.

[0081] It is well known that MMPs are expressed in many differenttissues, including the brain. Accordingly, the MMPs of the presentinvention may be useful, inter alia, for treating and/or diagnosingmental disorders. Following the techniques described in Example 4,below, those skilled in the art could readily ascertain if MMP isexpressed in a particular tissue or region.

[0082] The invention provides purified and isolated polynucleotides(e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof,whether single- or double-stranded) that comprise a nucleotide sequenceencoding the amino acid sequence of the polypeptides of the invention.Such polynucleotides are useful for recombinantly expressing the MMP andalso for detecting expression of the MMP in cells (e.g., using Northernhybridization and in situ hybridization assays). Such polynucleotidesalso are useful in the design of antisense and other molecules for thesuppression of the expression of MMP in a cultured cell, a tissue, or ananimal; for therapeutic purposes; or to provide a model for diseases orconditions characterized by aberrant MMP expression. Specificallyexcluded from the definition of polynucleotides of the invention areentire isolated, non-recombinant native chromosomes of host cells. Apreferred polynucleotide has a sequence selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:3, which correspond to naturallyoccurring MMP sequences. It will be appreciated that numerous otherpolynucleotide sequences exist that also encode MMP having the sequenceselected from the group consisting of SEQ ID NO:4 to SEQ ID NO:6, due tothe well-known degeneracy of the universal genetic code.

[0083] The invention also provides a purified and isolatedpolynucleotide comprising a nucleotide sequence that encodes a mammalianpolypeptide, wherein the polynucleotide hybridizes to a polynucleotidehaving the sequence set forth in sequences selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:3, or the non-coding strandcomplementary thereto, under the following hybridization conditions:

[0084] (a) hybridization for 16 hours at 42° C. in a hybridizationsolution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextransulfate; and

[0085] (b) washing 2 times for 30 minutes each at 60° C. in a washsolution comprising 0.1% SSC, 1% SDS. Polynucleotides that encode ahuman allelic variant are highly preferred.

[0086] The present invention relates to molecules which comprise thegene sequences that encode the MMPs; constructs and recombinant hostcells incorporating the gene sequences; the novel MMP polypeptidesencoded by the gene sequences; antibodies to the polypeptides andhomologs; kits employing the polynucleotides and polypeptides, andmethods of making and using all of the foregoing. In addition, thepresent invention relates to homologs of the gene sequences and of thepolypeptides and methods of making and using the same.

[0087] Genomic DNA of the invention comprises the protein-coding regionfor a polypeptide of the invention and is also intended to includeallelic variants thereof. It is widely understood that, for many genes,genomic DNA is transcribed into RNA transcripts that undergo one or moresplicing events wherein intron (i.e., non-coding regions) of thetranscripts are removed, or “spliced out.” RNA transcripts that can bespliced by alternative mechanisms, and therefore be subject to removalof different RNA sequences but still encode an MMP polypeptide, arereferred to in the art as splice variants which are embraced by theinvention. Splice variants comprehended by the invention therefore areencoded by the same original genomic DNA sequences but arise fromdistinct mRNA transcripts. Allelic variants are modified forms of awild-type gene sequence, the modification resulting from recombinationduring chromosomal segregation or exposure to conditions which give riseto genetic mutation. Allelic variants, like wild type genes, arenaturally occurring sequences (as opposed to non-naturally occurringvariants that arise from in vitro manipulation).

[0088] The invention also comprehends cDNA that is obtained throughreverse transcription of an RNA polynucleotide encoding MMP(conventionally followed by second strand synthesis of a complementarystrand to provide a double-stranded DNA).

[0089] Preferred DNA sequences encoding human MMP polypeptides areselected from the group consisting of SEQ ID NO:1 to SEQ ID NO:3. Apreferred DNA of the invention comprises a double stranded moleculealong with the complementary molecule (the “non-coding strand” or“complement”) having a sequence unambiguously deducible from the codingstrand according to Watson-Crick base-pairing rules for DNA. Alsopreferred are other polynucleotides encoding the MMP polypeptideselected from the group consisting of SEQ ID NO:4 to SEQ ID NO:6, whichdiffer in sequence from the polynucleotides selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:3, by virtue of the well-knowndegeneracy of the universal nuclear genetic code.

[0090] The invention further embraces other species, preferablymammalian, homologs of the human MMP DNA. Species homologs, sometimesreferred to as “orthologs,” in general, share at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% homology with human DNA of theinvention. Generally, percent sequence “homology” with respect topolynucleotides of the invention may be calculated as the percentage ofnucleotide bases in the candidate sequence that are identical tonucleotides in the MMP sequences set forth in sequences selected fromthe group consisting of SEQ ID NO:1 to SEQ ID NO:3, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity.

[0091] Polynucleotides of the invention permit identification andisolation of polynucleotides encoding related MMP polypeptides, such ashuman allelic variants and species homologs, by well-known techniquesincluding Southern and/or Northern hybridization, and polymerase chainreaction (PCR). Examples of related polynucleotides include human andnon-human genomic sequences, including allelic variants, as well aspolynucleotides encoding polypeptides homologous to MMP and structurallyrelated polypeptides sharing one or more biological, immunological,and/or physical properties of MMP. Non-human species genes encodingproteins homologous to MMP can also be identified by Southern and/or PCRanalysis and are useful in animal models for MMP disorders. Knowledge ofthe sequence of a human MMP DNA also makes possible through use ofSouthern hybridization or polymerase chain reaction (PCR) theidentification of genomic DNA sequences encoding expression controlregulatory sequences such as promoters, operators, enhancers,repressors, and the like. Polynucleotides of the invention are alsouseful in hybridization assays to detect the capacity of cells toexpress MMP. Polynucleotides of the invention may also provide a basisfor diagnostic methods useful for identifying a genetic alteration(s) inan MMP locus that underlies a disease state or states, which informationis useful both for diagnosis and for selection of therapeuticstrategies.

[0092] According to the present invention, the MMP nucleotide sequencesdisclosed herein may be used to identify homologs of the MMP, in otheranimals, including but not limited to humans and other mammals, andinvertebrates. Any of the nucleotide sequences disclosed herein, or anyportion thereof, can be used, for example, as probes to screen databasesor nucleic acid libraries, such as, for example, genomic or cDNAlibraries, to identify homologs, using screening procedures well knownto those skilled in the art. Accordingly, homologs having at least 50%,more preferably at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, more preferablyat least 95%, and most preferably at least 100% homology with MMPsequences can be identified.

[0093] The disclosure herein of full-length polynucleotides encoding MMPpolypeptides makes readily available to the worker of ordinary skill inthe art every possible fragment of the full-length polynucleotide.

[0094] In a preferred embodiment, the isolated nucleic acid comprises anucleotide sequence of SEQ ID NO: 2, and fragments thereof, that encodea polypeptide having a sequence of SEQ ID NO: 5, or fragments thereof.In a more preferred embodiment, the nucleotide is not SEQ ID NO:7 anddoes not encode a polypeptide with a sequence of SEQ ID NO:8.

[0095] As used in the present invention, fragments of MMP-encodingpolynucleotides comprise at least 10, and preferably at least 12, 14,16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotideencoding MMP. Preferably, fragment polynucleotides of the inventioncomprise sequences unique to the MMP-encoding polynucleotide sequence,and therefore hybridize under highly stringent or moderately stringentconditions only (i.e., “specifically”) to polynucleotides encoding MMP(or fragments thereof). Polynucleotide fragments of genomic sequences ofthe invention comprise not only sequences unique to the coding region,but also include fragments of the full-length sequence derived fromintrons, regulatory regions, and/or other non-translated sequences.Sequences unique to polynucleotides of the invention are recognizablethrough sequence comparison to other known polynucleotides, and can beidentified through use of alignment programs routinely utilized in theart, e.g., those made available in public sequence databases. Suchsequences also are recognizable from Southern hybridization analyses todetermine the number of fragments of genomic DNA to which apolynucleotide will hybridize. Polynucleotides of the invention can belabeled in a manner that permits their detection, including radioactive,fluorescent, and enzymatic labeling.

[0096] Fragment polynucleotides are particularly useful as probes fordetection of full-length or fragments of MMP polynucleotides. One ormore polynucleotides can be included in kits that are used to detect thepresence of a polynucleotide encoding MMP, or used to detect variationsin a polynucleotide sequence encoding MMP.

[0097] The invention also embraces DNAs encoding MMP polypeptides thathybridize under moderately stringent or high stringency conditions tothe non-coding strand, or complement, of the polynucleotides set forthin sequences selected from the group consisting of SEQ ID NO:1 to SEQ IDNO:3.

[0098] Exemplary highly stringent hybridization conditions are asfollows: hybridization at 42° C. in a hybridization solution comprising50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twicefor 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1%SDS. It is understood in the art that conditions of equivalentstringency can be achieved through variation of temperature and buffer,or salt concentration as described Ausubel et al. (Eds.), Protocols inMolecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10.Modifications in hybridization conditions can be empirically determinedor precisely calculated based on the length and the percentage ofguanosine/cytosine (GC) base pairing of the probe. The hybridizationconditions can be calculated as described in Sambrook, et al., (Eds.),Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.

[0099] With the knowledge of the nucleotide sequence informationdisclosed in the present invention, one skilled in the art can identifyand obtain nucleotide sequences which encode MMP from different sources(i.e., different tissues or different organisms) through a variety ofmeans well known to the skilled artisan and as disclosed by, forexample, Sambrook et al., “Molecular cloning: a laboratory manual”,Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989), which is incorporated herein by reference in its entirety.

[0100] For example, DNA that encodes MMP may be obtained by screening ofmRNA, cDNA, or genomic DNA with oligonucleotide probes generated fromthe MMP gene sequence information provided herein. Probes may be labeledwith a detectable group, such as a fluorescent group, a radioactive atomor a chemiluminescent group in accordance with procedures known to theskilled artisan and used in conventional hybridization assays, asdescribed by, for example, Sambrook et al.

[0101] A nucleic acid molecule comprising any of the MMP nucleotidesequences described above can alternatively be synthesized by use of thepolymerase chain reaction (PCR) procedure, with the PCR oligonucleotideprimers produced from the nucleotide sequences provided herein. See U.S.Pat. No. 4,683,195 to Mullis et al. and 4,683,202 to Mullis. The PCRreaction provides a method for selectively increasing the concentrationof a particular nucleic acid sequence even when that sequence has notbeen previously purified and is present only in a single copy in aparticular sample. The method can be used to amplify either single- ordouble-stranded DNA. The essence of the method involves the use of twooligonucleotide probes to serve as primers for the template-dependent,polymerase mediated replication of a desired nucleic acid molecule.

[0102] A wide variety of alternative cloning and in vitro amplificationmethodologies are well known to those skilled in the art. Examples ofthese techniques are found in, for example, Berger et al., Guide toMolecular Cloning Techniques, Methods in Enzymology 152, Academic Press,Inc., San Diego, Calif. (Berger), which is incorporated herein byreference in its entirety.

[0103] Automated sequencing methods can be used to obtain or verify thenucleotide sequence of MMP. The MMP nucleotide sequences of the presentinvention are believed to be 100% accurate. However, as is known in theart, nucleotide sequence obtained by automated methods may contain someerrors. Nucleotide sequences determined by automation are typically atleast about 90%, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of a given nucleicacid molecule. The actual sequence may be more precisely determinedusing manual sequencing methods, which are well known in the art. Anerror in a sequence which results in an insertion or deletion of one ormore nucleotides may result in a frame shift in translation such thatthe predicted amino acid sequence will differ from that which would bepredicted from the actual nucleotide sequence of the nucleic acidmolecule, starting at the point of the mutation.

[0104] The nucleic acid molecules of the present invention, andfragments derived therefrom, are useful for screening for restrictionfragment length polymorphism (RFLP) associated with certain disorders,as well as for genetic mapping.

[0105] The polynucleotide sequence information provided by the inventionmakes possible large-scale expression of the encoded polypeptide bytechniques well known and routinely practiced in the art.

[0106] Vectors

[0107] Another aspect of the present invention is directed to vectors,or recombinant expression vectors, comprising any of the nucleic acidmolecules described above. Vectors are used herein either to amplify DNAor RNA encoding MMP and/or to express DNA which encodes MMP. Preferredvectors include, but are not limited to, plasmids, phages, cosmids,episomes, viral particles or viruses, and integratable DNA fragments(i.e., fragments integratable into the host genome by homologousrecombination). Preferred viral particles include, but are not limitedto, adenoviruses, baculoviruses, parvoviruses, herpesviruses,poxviruses, adeno-associated viruses, Semliki Forest viruses, vacciniaviruses, and retroviruses. Preferred expression vectors include, but arenot limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). Otherexpression vectors include, but are not limited to, pSPORT™ vectors,pGEM™ vectors (Promega), pPROEXvectors™ (LTI, Bethesda, Md.),Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™(Invitrogen), and pYES2™(Invitrogen).

[0108] Expression constructs preferably comprise MMP-encodingpolynucleotides operatively linked to an endogenous or exogenousexpression control DNA sequence and a transcription terminator.Expression control DNA sequences include promoters, enhancers,operators, and regulatory element binding sites generally, and aretypically selected based on the expression systems in which theexpression construct is to be utilized. Preferred promoter and enhancersequences are generally selected for the ability to increase geneexpression, while operator sequences are generally selected for theability to regulate gene expression. Expression constructs of theinvention may also include sequences encoding one or more selectablemarkers that permit identification of host cells bearing the construct.Expression constructs may also include sequences that facilitate, andpreferably promote, homologous recombination in a host cell. Preferredconstructs of the invention also include sequences necessary forreplication in a host cell.

[0109] Expression constructs are preferably utilized for production ofan encoded protein, but may also be utilized simply to amplify anMMP-encoding polynucleotide sequence. In preferred embodiments, thevector is an expression vector wherein the polynucleotide of theinvention is operatively linked to a polynucleotide comprising anexpression control sequence. Autonomously replicating recombinantexpression constructs such as plasmid and viral DNA vectorsincorporating polynucleotides of the invention are also provided.Preferred expression vectors are replicable DNA constructs in which aDNA sequence encoding MMP is operably linked or connected to suitablecontrol sequences capable of effecting the expression of the MMP in asuitable host. DNA regions are operably linked or connected when theyare functionally related to each other. For example, a promoter isoperably linked or connected to a coding sequence if it controls thetranscription of the sequence. Amplification vectors do not requireexpression control domains, but rather need only the ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants. The needfor control sequences in the expression vector will vary depending uponthe host selected and the transformation method chosen. Generally,control sequences include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding and sequences which control the termination oftranscription and translation.

[0110] Preferred vectors preferably contain a promoter that isrecognized by the host organism. The promoter sequences of the presentinvention may be prokaryotic, eukaryotic or viral. Examples of suitableprokaryotic sequences include the P_(R) and P_(L) promoters ofbacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1973), which isincorporated herein by reference in its entirety; Lambda II, Hendrix, R.W., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1980),which is incorporated herein by reference in its entirety); the trp,recA, heat shock, and lacZ promoters of E. coli and the SV40 earlypromoter (Benoist et al. Nature, 1981, 290, 304-310, which isincorporated herein by reference in its entirety). Additional promotersinclude, but are not limited to, mouse mammary tumor virus, longterminal repeat of human immunodeficiency virus, maloney virus,cytomegalovirus immediate early promoter, Epstein Barr virus, Roussarcoma virus, human actin, human myosin, human hemoglobin, human musclecreatine, and human metalothionein.

[0111] Additional regulatory sequences can also be included in preferredvectors. Preferred examples of suitable regulatory sequences arerepresented by the Shine-Dalgamo of the replicase gene of the phage MS-2and of the gene cII of bacteriophage lambda. The Shine-Dalgarno sequencemay be directly followed by DNA encoding MMP and result in theexpression of the mature MMP protein.

[0112] Moreover, suitable expression vectors can include an appropriatemarker that allows the screening of the transformed host cells. Thetransformation of the selected host is carried out using any one of thevarious techniques well known to the expert in the art and described inSambrook et al., supra.

[0113] An origin of replication can also be provided either byconstruction of the vector to include an exogenous origin or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter may besufficient. Alternatively, rather than using vectors which contain viralorigins of replication, one skilled in the art can transform mammaliancells by the method of co-transformation with a selectable marker andMMP DNA. An example of a suitable marker is dihydrofolate reductase(DHFR) or thymidine kinase (see, U.S. Pat. No. 4,399,216).

[0114] Nucleotide sequences encoding MMP may be recombined with vectorDNA in accordance with conventional techniques, including blunt-ended orstaggered-ended termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesiderable joining, andligation with appropriate ligases. Techniques for such manipulation aredisclosed by Sambrook et al., supra and are well known in the art.Methods for construction of mammalian expression vectors are disclosedin, for example, Okayama et al., Mol. Cell. Biol., 1983, 3, 280, Cosmanet al., Mol. Immunol., 1986, 23, 935, Cosman et al., Nature, 1984, 312,768, EP-A-0367566, and WO 91/18982, each of which is incorporated hereinby reference in its entirety.

[0115] Host Cells

[0116] According to another aspect of the invention, host cells areprovided, including prokaryotic and eukaryotic cells, comprising apolynucleotide of the invention (or vector of the invention) in a mannerthat permits expression of the encoded MMP polypeptide. Polynucleotidesof the invention may be introduced into the host cell as part of acircular plasmid, or as linear DNA comprising an isolated protein codingregion or a viral vector. Methods for introducing DNA into the host cellthat are well known and routinely practiced in the art includetransformation, transfection, electroporation, nuclear injection, orfusion with carriers such as liposomes, micelles, ghost cells, andprotoplasts. Expression systems of the invention include bacterial,yeast, fungal, plant, insect, invertebrate, vertebrate, and mammaliancells systems.

[0117] The invention provides host cells that are transformed ortransfected (stably or transiently) with polynucleotides of theinvention or vectors of the invention. As stated above, such host cellsare useful for amplifying the polynucleotides and also for expressingthe MMP polypeptide or fragment thereof encoded by the polynucleotide.

[0118] In still another related embodiment, the invention provides amethod for producing a MMP polypeptide (or fragment thereof) comprisingthe steps of growing a host cell of the invention in a nutrient mediumand isolating the polypeptide or variant thereof from the cell or themedium.

[0119] According to some aspects of the present invention, transformedhost cells having an expression vector comprising any of the nucleicacid molecules described above are provided. Expression of thenucleotide sequence occurs when the expression vector is introduced intoan appropriate host cell. Suitable host cells for expression of thepolypeptides of the invention include, but are not limited to,prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vectoris employed, then the appropriate host cell would be any prokaryoticcell capable of expressing the cloned sequences. Suitable prokaryoticcells include, but are not limited to, bacteria of the generaEscherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, andStaphylococcus.

[0120] If an eukaryotic expression vector is employed, then theappropriate host cell would be any eukaryotic cell capable of expressingthe cloned sequence. Preferably, eukaryotic cells are cells of highereukaryotes. Suitable eukaryotic cells include, but are not limited to,non-human mammalian tissue culture cells and human tissue culture cells.Preferred host cells include, but are not limited to, insect cells, HeLacells, Chinese hamster ovary cells (CHO cells), African green monkeykidney cells (COS cells), human HEK-293 cells, and murine 3T3fibroblasts. Propagation of such cells in cell culture has become aroutine procedure (see, Tissue Culture, Academic Press, Kruse andPatterson, eds. (1973), which is incorporated herein by reference in itsentirety).

[0121] In addition, a yeast host may be employed as a host cell.Preferred yeast cells include, but are not limited to, the generaSaccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S.cerevisiae and P. pastoris. Preferred yeast vectors can contain anorigin of replication sequence from a 2T yeast plasmid, an autonomouslyreplication sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Shuttle vectors for replication in both yeastand E. coli are also included herein.

[0122] Alternatively, insect cells may be used as host cells. In apreferred embodiment, the polypeptides of the invention are expressedusing a baculovirus expression system (see, Luckow et al.,Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: ALaboratory Manual, O'Rielly et al. (Eds.), W. H. Freeman and Company,New York, 1992, and U.S. Pat. No. 4,879,236, each of which isincorporated herein by reference in its entirety). In addition, theMAXBAC™ complete baculovirus expression system (Invitrogen) can, forexample, be used for production in insect cells.

[0123] Host cells of the invention are a valuable source of immunogenfor development of antibodies specifically immunoreactive with MMP. Hostcells of the invention are also useful in methods for the large-scaleproduction of MMP polypeptides wherein the cells are grown in a suitableculture medium and the desired polypeptide products are isolated fromthe cells, or from the medium in which the cells are grown, bypurification methods known in the art, e.g., conventionalchromatographic methods including immunoaffinity chromatography,affinity chromatography, hydrophobic interaction chromatography, lectinaffinity chromatography, size exclusion filtration, cation or anionexchange chromatography, high pressure liquid chromatography (HPLC),reverse phase HPLC, and the like. Still other methods of purificationinclude those methods wherein the desired protein is expressed andpurified as a fusion protein having a specific tag, label, or chelatingmoiety that is recognized by a specific binding partner or agent. Thepurified protein can be cleaved to yield the desired protein, or can beleft as an intact fusion protein. Cleavage of the fusion component mayproduce a form of the desired protein having additional amino acidresidues as a result of the cleavage process.

[0124] Knowledge of MMP DNA sequences allows for modification of cellsto permit, or increase, expression of endogenous MMP. Cells can bemodified (e.g., by homologous recombination) to provide increasedexpression by replacing, in whole or in part, the naturally occurringMMP promoter with all or part of a heterologous promoter so that thecells express MMP at higher levels. The heterologous promoter isinserted in such a manner that it is operatively linked to endogenousMMP encoding sequences. (See, for example, PCT International PublicationNo. WO 94/12650, PCT International Publication No. WO 92/20808, and PCTInternational Publication No. WO 91/09955.) It is also contemplatedthat, in addition to heterologous promoter DNA, amplifiable marker DNA(e.g., ada, dhfr, and the multifunctional CAD gene which encodescarbamoyl phosphate synthase, aspartate transcarbamylase, anddihydroorotase) and/or intron DNA may be inserted along with theheterologous promoter DNA. If linked to the MMP coding sequence,amplification of the marker DNA by standard selection methods results inco-amplification of the MMP coding sequences in the cells.

[0125] Knock-outs

[0126] The DNA sequence information provided by the present inventionalso makes possible the development (e.g., by homologous recombinationor “knock-out” strategies; see Capecchi, Science 244:1288-1292 (1989),which is incorporated herein by reference) of animals that fail toexpress functional MMP or that express a variant of MMP. Such animals(especially small laboratory animals such as rats, rabbits, and mice)are useful as models for studying the in vivo activities of MMP andmodulators of MMP.

[0127] Antisense

[0128] Also made available by the invention are anti-sensepolynucleotides that recognize and hybridize to polynucleotides encodingNMP. Full-length and fragment anti-sense polynucleotides are provided.Fragment antisense molecules of the invention include (i) those thatspecifically recognize and hybridize to MMP RNA (as determined bysequence comparison of DNA encoding MMP to DNA encoding other knownmolecules). Identification of sequences unique to MMP encodingpolynucleotides can be deduced through use of any publicly availablesequence database, and/or through use of commercially available sequencecomparison programs. After identification of the desired sequences,isolation through restriction digestion or amplification using any ofthe various polymerase chain reaction techniques well known in the artcan be performed. Anti-sense polynucleotides are particularly relevantto regulating expression of MMP by those cells expressing MMP mRNA.

[0129] Antisense nucleic acids (preferably 10 to 30 base-pairoligonucleotides) capable of specifically binding to MMP expressioncontrol sequences or MMP RNA are introduced into cells (e.g., by a viralvector or colloidal dispersion system such as a liposome). The antisensenucleic acid binds to the MMP target nucleotide sequence in the cell andprevents transcription and/or translation of the target sequence.Phosphorothioate and methylphosphonate antisense oligonucleotides arespecifically contemplated for therapeutic use by the invention. Theantisense oligonucleotides may be further modified by addingpoly-L-lysine, transferrin polylysine, or cholesterol moieties at their5′ end. Suppression of MMP expression at either the transcriptional ortranslational level is useful to generate cellular or animal models fordiseases/conditions characterized by aberrant MMP expression.

[0130] Antisense oligonucleotides, or fragments of sequences selectedfrom the group consisting of SEQ ID NO:1 to SEQ ID NO:3, or sequencescomplementary or homologous thereto, derived from the nucleotidesequences of the present invention encoding MMP are useful as diagnostictools for probing gene expression in various tissues. For example,tissue can be probed in situ with oligonucleotide probes carryingdetectable groups by conventional autoradiography techniques toinvestigate native expression of this enzyme or pathological conditionsrelating thereto. Antisense oligonucleotides are preferably directed toregulatory regions of sequences selected from the group consisting ofSEQ ID NO:1 to SEQ ID NO:3, or mRNA corresponding thereto, including,but not limited to, the initiation codon, TATA box, enhancer sequences,and the like.

[0131] Transcription Factors

[0132] The MMP sequences taught in the present invention facilitate thedesign of novel transcription factors for modulating MMP expression innative cells and animals, and cells transformed or transfected with MMPpolynucleotides. For example, the Cys₂-His₂ zinc finger proteins, whichbind DNA via their zinc finger domains, have been shown to be amenableto structural changes that lead to the recognition of different targetsequences. These artificial zinc finger proteins recognize specifictarget sites with high affinity and low dissociation constants, and areable to act as gene switches to modulate gene expression. Knowledge ofthe particular MMP target sequence of the present invention facilitatesthe engineering of zinc finger proteins specific for the target sequenceusing known methods such as a combination of structure-based modelingand screening of phage display libraries (Segal et al., Proc. Natl.Acad. Sci. (USA) 96:2758-2763 (1999); Liu et al., Proc. Natl. Acad. Sci.(USA) 94:5525-5530 (1997); Greisman et al., Science 275:657-661 (1997);Choo et al., J. Mol. Biol. 273:525-532 (1997)). Each zinc finger domainusually recognizes three or more base pairs. Since a recognitionsequence of 18 base pairs is generally sufficient in length to render itunique in any known genome, a zinc finger protein consisting of 6 tandemrepeats of zinc fingers would be expected to ensure specificity for aparticular sequence (Segal et al.) The artificial zinc finger repeats,designed based on MMP sequences, are fused to activation or repressiondomains to promote or suppress MMP expression (Liu et al.)Alternatively, the zinc finger domains can be fused to the TATAbox-binding factor (TBP) with varying lengths of linker region betweenthe zinc finger peptide and the TBP to create either transcriptionalactivators or repressors (Kim et al., Proc. Natl. Acad. Sci. (USA)94:3616-3620 (1997). Such proteins and polynucleotides that encode them,have utility for modulating MMP expression in vivo in both native cells,animals and humans; and/or cells transfected with MMP-encodingsequences. The novel transcription factor can be delivered to the targetcells by transfecting constructs that express the transcription factor(gene therapy), or by introducing the protein. Engineered zinc fingerproteins can also be designed to bind RNA sequences for use intherapeutics as alternatives to antisense or catalytic RNA methods(McColl et al., Proc. Natl. Acad. Sci. (USA) 96:9521-9526 (1997); Wu etal., Proc. Natl. Acad. Sci. (USA) 92:344-348 (1995)). The presentinvention contemplates methods of designing such transcription factorsbased on the gene sequence of the invention, as well as customized zincfinger proteins, that are useful to modulate MMP expression in cells(native or transformed) whose genetic complement includes thesesequences.

[0133] Polypeptides

[0134] The invention also provides purified and isolated mammalian MMPpolypeptides encoded by a polynucleotide of the invention. Presentlypreferred is a human MMP polypeptide comprising the amino acid sequenceset out in sequences selected from the group consisting of SEQ ID NO:4to SEQ ID NO:6, or fragments thereof comprising an epitope specific tothe polypeptide. By “epitope specific to” is meant a portion of the MMPthat is recognizable by an antibody that is specific for the MMP, asdefined in detail below.

[0135] Although the sequences provided are particular human sequences,the invention is intended to include within its scope other humanallelic variants; non-human mammalian forms of MMP, and other vertebrateforms of MMP.

[0136] The invention also embraces polypeptides that have at least 99%,at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55% or at least 50%identity and/or homology to the preferred polypeptide of the invention.Percent amino acid sequence “identity” with respect to the preferredpolypeptide of the invention is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical withthe residues in the MMP sequence after aligning both sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Percent sequence “homology” with respect to thepreferred polypeptide of the invention is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the residues in the MMP sequence after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and also considering any conservativesubstitutions as part of the sequence identity.

[0137] In one aspect, percent homology is calculated as the percentageof amino acid residues in the smaller of two sequences which align withidentical amino acid residue in the sequence being compared, when fourgaps in a length of 100 amino acids may be introduced to maximizealignment (Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5,p. 124, National Biochemical Research Foundation, Washington, D.C.(1972), incorporated herein by reference).

[0138] Polypeptides of the invention may be isolated from natural cellsources or may be chemically synthesized, but are preferably produced byrecombinant procedures involving host cells of the invention. Use ofmammalian host cells is expected to provide for such post-translationalmodifications (e.g., glycosylation, truncation, lipidation, andphosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention. Glycosylated andnon-glycosylated forms of MMP polypeptides are embraced by theinvention.

[0139] The invention also embraces variant (or analog) MMP polypeptides.In one example, insertion variants are provided wherein one or moreamino acid residues supplement a MMP amino acid sequence. Insertions maybe located at either or both termini of the protein, or may bepositioned within internal regions of the MMP amino acid sequence.Insertional variants with additional residues at either or both terminican include, for example, fusion proteins and proteins including aminoacid tags or labels.

[0140] Insertion variants include MMP polypeptides wherein one or moreamino acid residues are added to a MMP acid sequence or to abiologically active fragment thereof.

[0141] Variant products of the invention also include mature MMPproducts, i.e., MMP products wherein leader or signal sequences areremoved, with additional amino terminal residues. The additional aminoterminal residues may be derived from another protein, or may includeone or more residues that are not identifiable as being derived fromspecific proteins. MMP products with an additional methionine residue atposition −1 (Met⁻¹-MMP) are contemplated, as are variants withadditional methionine and lysine residues at positions −2 and −1(Met⁻²-Lys⁻¹-MMP). Variants of MMP with additional Met, Met-Lys, Lysresidues (or one or more basic residues in general) are particularlyuseful for enhanced recombinant protein production in bacterial hostcells.

[0142] The invention also embraces MMP variants having additional aminoacid residues that result from use of specific expression systems. Forexample, use of commercially available vectors that express a desiredpolypeptide as part of a glutathione-S-transferase (GST) fusion productprovides the desired polypeptide having an additional glycine residue atposition −1 after cleavage of the GST component from the desiredpolypeptide. Variants that result from expression in other vectorsystems are also contemplated.

[0143] Insertional variants also include fusion proteins wherein theamino terminus and/or the carboxy terminus of MMP is/are fused toanother polypeptide.

[0144] In another aspect, the invention provides deletion variantswherein one or more amino acid residues in a MMP polypeptide areremoved. Deletions can be effected at one or both termini of the MMPpolypeptide, or with removal of one or more non-terminal amino acidresidues of MMP. Deletion variants, therefore, include all fragments ofa MMP polypeptide.

[0145] The invention also embraces polypeptide fragments of sequencesselected from the group consisting of SEQ ID NO:4 to SEQ ID NO:6,wherein the fragments maintain biological (e.g., proteinase orcollagenase activity) and immunological properties of a MMP polypeptide.

[0146] In one preferred embodiment of the invention, an isolated nucleicacid molecule comprises a nucleotide sequence that encodes a polypeptidecomprising an amino acid sequence homologous to sequences selected fromthe group consisting of SEQ ID NO:4 to SEQ ID NO:6, and fragmentsthereof, wherein the nucleic acid molecule encoding at least a portionof MMP. In a more preferred embodiment, the isolated nucleic acidmolecule comprises a sequence that encodes a polypeptide comprisingsequences selected from the group consisting of SEQ ID NO:4 to SEQ EDNO:6, and fragments thereof.

[0147] One preferred embodiment of the present invention provides anisolated nucleic acid molecule comprising a sequence homologoussequences selected from the group consisting of SEQ ID NO: 1 to SEQ IDNO:3, and fragments thereof. Another preferred embodiment provides anisolated nucleic acid molecule comprising a sequence selected from thegroup consisting of SEQ ID NO:1 to SEQ ID NO:3, and fragments thereof.

[0148] In a preferred embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence which encodes a polypeptide comprising asequence of SEQ ID NO:5, or a fragment thereof. In a more preferredembodiment, the polypeptide encoded by the nucleotide sequence does nothave the sequence of SEQ ID NO:8.

[0149] As used in the present invention, polypeptide fragments compriseat least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids ofsequences selected from the group consisting of SEQ ID NO:4 to SEQ IDNO:6. Preferred polypeptide fragments display antigenic propertiesunique to, or specific for, human MMP and its allelic and specieshomologs. Fragments of the invention having the desired biological andimmunological properties can be prepared by any of the methods wellknown and routinely practiced in the art.

[0150] In still another aspect, the invention provides substitutionvariants of MMP polypeptides. Substitution variants include thosepolypeptides wherein one or more amino acid residues of a MMPpolypeptide are removed and replaced with alternative residues. In oneaspect, the substitutions are conservative in nature; however, theinvention embraces substitutions that are also non-conservative.Conservative substitutions for this purpose may be defined as set out inTables 2, 3, or 4 below.

[0151] Variant polypeptides include those wherein conservativesubstitutions have been introduced by modification of polynucleotidesencoding polypeptides of the invention. Amino acids can be classifiedaccording to physical properties and contribution to secondary andtertiary protein structure. A conservative substitution is recognized inthe art as a substitution of one amino acid for another amino acid thathas similar properties. Exemplary conservative substitutions are set outin Table 2 (from WO 97/09433, page 10, published Mar. 13, 1997(PCT/GB96/02197, filed Sep. 6, 1996), immediately below. TABLE 2Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINO ACIDAliphatic Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R Aromatic H F W Y Other N Q D E

[0152] Alternatively, conservative amino acids can be grouped asdescribed in Lehninger, [Biochemistry, Second Edition; Worth Publishers,Inc. NY, N.Y. (1975), pp.71-77] as set out in Table 3, below. TABLE 3Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINO ACIDNon-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D E

[0153] As still another alternative, exemplary conservativesubstitutions are set out in Table 4, below. TABLE 4 ConservativeSubstitutions III Original Residue Exemplary Substitution Ala (A) Val,Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) GluCys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I)Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg,Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) GlySer (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V)Ile, Leu, Met, Phe, Ala

[0154] It should be understood that the definition of polypeptides ofthe invention is intended to include polypeptides bearing modificationsother than insertion, deletion, or substitution of amino acid residues.By way of example, the modifications may be covalent in nature, andinclude for example, chemical bonding with polymers, lipids, otherorganic, and inorganic moieties. Such derivatives may be prepared toincrease circulating half-life of a polypeptide, or may be designed toimprove the targeting capacity of the polypeptide for desired cells,tissues, or organs. Similarly, the invention further embraces MMPpolypeptides that have been covalently modified to include one or morewater-soluble polymer attachments such as polyethylene glycol,polyoxyethylene glycol, or polypropylene glycol. Variants that displayligand binding properties of native MMP and are expressed at higherlevels are particularly useful in assays of the invention; the variantsare also useful in providing cellular, tissue and animal models ofdiseases/conditions characterized by aberrant MMP activity.

[0155] In a related embodiment, the present invention providescompositions comprising purified polypeptides of the invention.Preferred compositions comprise, in addition to the polypeptide of theinvention, a pharmaceutically acceptable (i.e., sterile and non-toxic)liquid, semisolid, or solid diluent that serves as a pharmaceuticalvehicle, excipient, or medium. Any diluent known in the art may be used.Exemplary diluents include, but are not limited to, water, salinesolutions, polyoxyethylene sorbitan monolaurate, magnesium stearate,methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose,sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate,mineral oil, and cocoa butter.

[0156] Antibodies

[0157] Also comprehended by the present invention are antibodies (e.g.,monoclonal and polyclonal antibodies, single chain antibodies, chimericantibodies, bifunctional/bispecific antibodies, humanized antibodies,human antibodies, and complementary determining region (CDR)-graftedantibodies, including compounds which include CDR sequences whichspecifically recognize a polypeptide of the invention) specific for MMPor fragments thereof. Preferred antibodies of the invention are humanantibodies that are produced and identified according to methodsdescribed in WO93/11236, published Jun. 20, 1993, which is incorporatedherein by reference in its entirety. Antibody fragments, including Fab,Fab′, F(ab′)₂, and F_(v), are also provided by the invention. The term“specific for,” when used to describe antibodies of the invention,indicates that the variable regions of the antibodies of the inventionrecognize and bind MMP polypeptides exclusively (i.e., are able todistinguish MMP polypeptides from other known MMP polypeptides by virtueof measurable differences in binding affinity, despite the possibleexistence of localized sequence identity, homology, or similaritybetween MMP and such polypeptides). It will be understood that specificantibodies may also interact with other proteins (for example, S. aureusprotein A or other antibodies in ELISA techniques) through interactionswith sequences outside the variable region of the antibodies, and, inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of an antibody of the invention are wellknown and routinely practiced in the art. For a comprehensive discussionof such assays, see Harlow et al. (Eds.), Antibodies A LaboratoryManual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988),Chapter 6. Antibodies that recognize and bind fragments of the MMPpolypeptides of the invention are also contemplated, provided that theantibodies are specific for MMP polypeptides. Antibodies of theinvention can be produced using any method well known and routinelypracticed in the art.

[0158] The invention provides an antibody that is specific for the MMPof the invention. Antibody specificity is described in greater detailbelow. However, it should be emphasized that antibodies that can begenerated from polypeptides that have previously been described in theliterature and that are capable of fortuitously cross-reacting with MMP(e.g., due to the fortuitous existence of a similar epitope in bothpolypeptides) are considered “cross-reactive” antibodies. Suchcross-reactive antibodies are not antibodies that are “specific” forMMP. The determination of whether an antibody is specific for MMP or iscross-reactive with another known MMP is made using any of severalassays, such as Western blotting assays, that are well known in the art.

[0159] In one preferred variation, the invention provides monoclonalantibodies. Hybridomas that produce such antibodies also are intended asaspects of the invention. In yet another variation, the inventionprovides a humanized antibody. Humanized antibodies are useful for invivo therapeutic indications.

[0160] In another variation, the invention provides a cell-freecomposition comprising polyclonal antibodies, wherein at least one ofthe antibodies is an antibody of the invention specific for MMP.Antisera isolated from an animal is an exemplary composition, as is acomposition comprising an antibody fraction of an antisera that has beenresuspended in water or in another diluent, excipient, or carrier.

[0161] In still another related embodiment, the invention provides ananti-idiotypic antibody specific for an antibody that is specific forMMP.

[0162] It is well known that antibodies contain relatively small antigenbinding domains that can be isolated chemically or by recombinanttechniques. Such domains are useful MMP binding molecules themselves,and also may be reintroduced into human antibodies, or fused to toxinsor other polypeptides. Thus, in still another embodiment, the inventionprovides a polypeptide comprising a fragment of an MMP-specificantibody, wherein the fragment and the polypeptide bind to the MMP. Byway of non-limiting example, the invention provides polypeptides thatare single chain antibodies and CDR-grafted antibodies.

[0163] Non-human antibodies may be humanized by any of the methods knownin the art. In one method, the non-human CDRs are inserted into a humanantibody or consensus antibody framework sequence. Further changes canthen be introduced into the antibody framework to modulate affinity orimmunogenicity.

[0164] Antibodies of the invention are useful for, e.g., therapeuticpurposes (by modulating activity of MMP), diagnostic purposes to detector quantitate MMP, and purification of MMP. Kits comprising an antibodyof the invention for any of the purposes described herein are alsocomprehended. In general, a kit of the invention also includes a controlantigen for which the antibody is immunospecific.

[0165] Compositions

[0166] Mutations in the MMP gene that result in loss of normal functionof the MMP gene product underlie MMP-related human disease states. Theinvention comprehends gene therapy to restore MMP activity to treatthose disease states. Delivery of a functional MMP gene to appropriatecells is effected ex vivo, in situ, or in vivo by use of vectors, andmore particularly viral vectors (e.g., adenovirus, adeno-associatedvirus, or a retrovirus), or ex vivo by use of physical DNA transfermethods (e.g., liposomes or chemical treatments). See, for example,Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). Foradditional reviews of gene therapy technology see Friedmann, Science,244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); andMiller, Nature, 357: 455-460 (1992). Alternatively, it is contemplatedthat in other human disease states, preventing the expression of, orinhibiting the activity of, MMP will be useful in treating diseasestates. It is contemplated that antisense therapy or gene therapy couldbe applied to negatively regulate the expression of MMP.

[0167] Another aspect of the present invention is directed tocompositions, including pharmaceutical compositions, comprising any ofthe nucleic acid molecules or recombinant expression vectors describedabove and an acceptable carrier or diluent. Preferably, the carrier ordiluent is pharmaceutically acceptable. Suitable carriers are describedin the most recent edition of Remington's Pharnaceutical Sciences, A.Osol, a standard reference text in this field, which is incorporatedherein by reference in its entirety. Preferred examples of such carriersor diluents include, but are not limited to, water, saline, Ringer'ssolution, dextrose solution, and 5% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils may also be used. Theformulations are sterilized by commonly used techniques.

[0168] Also within the scope of the invention are compositionscomprising polypeptides, polynucleotides, or antibodies of the inventionthat have been formulated with, e.g., a pharmaceutically acceptablecarrier.

[0169] The invention also provides methods of using antibodies of theinvention. For example, the invention provides a method for modulatingligand binding of a MMP comprising the step of contacting the MMP withan antibody specific for the MMP, under conditions wherein the antibodybinds the MMP.

[0170] As discussed above, it is well known that MMPs are expressed inmany different tissues and regions, including in the brain. MMPs thatmay be expressed in the brain provide an indication that aberrant MMPactivity may correlate with one or more neurological or psychologicaldisorders. The invention also provides a method for treating aneurological or psychiatric disorder comprising the step ofadministering to a mammal in need of such treatment an amount of anantibody-like polypeptide of the invention that is sufficient tomodulate ligand binding to a MMP in neurons of the mammal. MMP may alsobe expressed in other tissues, including but not limited to, includingbut not limited to pancreas (and particularly pancreatic islet tissue),pituitary, skeletal muscle, adipose tissue, liver, and thyroid, and maybe found in many other tissues.

[0171] Kits

[0172] The present invention is also directed to kits, includingpharmaceutical kits. The kits can comprise any of the nucleic acidmolecules described above, any of the polypeptides described above, orany antibody which binds to a polypeptide of the invention as describedabove, as well as a negative control. The kit preferably comprisesadditional components, such as, for example, instructions, solidsupport, reagents helpful for quantification, and the like.

[0173] In another aspect, the invention features methods for detectionof a polypeptide in a sample as a diagnostic tool for diseases ordisorders, wherein the method comprises the steps of: (a) contacting thesample with a nucleic acid probe which hybridizes under hybridizationassay conditions to a nucleic acid target region of a polypeptide havingsequences selected from the group consisting of SEQ ID NO:4 to SEQ IDNO:6, said probe comprising the nucleic acid sequence encoding thepolypeptide, fragments thereof, and the complements of the sequences andfragments; and (b) detecting the presence or amount of the probe:targetregion hybrid as an indication of the disease.

[0174] In preferred embodiments of the invention, the disease isselected from the group consisting of metabolic diseases and disorders(e.g., type 2 diabetes, obesity, cardiovascular, dyslipidemias,adipogenesis, retinopathies, neuropathies, nephropathies etc.),proliferative diseases and cancers (e.g., different cancers such asbreast, colon, lung, etc., tumor growth, tumor invasion, andhyperproliferative disorders such as psoriasis, prostate hyperplasia,etc.), hormonal disorders (e.g., male/female hormonal replacement,polycystic ovarian syndrome, alopecia, etc.), CNS disorders (e.g.,degenerative disorders such as Parkinson's, Alzheimer's, etc.),inflammatory conditions (e.g., Chron's disease, arthritis), diseasesrelated to cell differentiation and homeostasis, cardiomyopathy,atherosclerosis, thromboembolic diseases, Sjögren's syndrome, renalfailure, periodontal diseases, retinal neovascularization, woundhealing, and neurodegenerative diseases including, for example,Alzheimer's disease, multiple sclerosis, Parkinson's disease, andmotoneuron disease, among others.

[0175] Kits may be designed to detect either expression ofpolynucleotides encoding MMP expressed in the brain or the MMP proteinsthemselves in order to identify tissue as being neurological. Forexample, oligonucleotide hybridization kits can be provided whichinclude a container having an oligonucleotide probe specific for theMMP-specific DNA and optionally, containers with positive and negativecontrols and/or instructions. Similarly, PCR kits can be provided whichinclude a container having primers specific for the NMP-specificsequences, DNA and optionally, containers with size markers, positiveand negative controls and/or instructions.

[0176] Hybridization conditions should be such that hybridization occursonly with the genes in the presence of other nucleic acid molecules.Under stringent hybridization conditions only highly complementarynucleic acid sequences hybridize. Preferably, such conditions preventhybridization of nucleic acids having 1 or 2 mismatches out of 20contiguous nucleotides. Such conditions are defined supra.

[0177] The diseases for which detection of genes in a sample could bediagnostic include diseases in which nucleic acid (DNA and/or RNA) isamplified in comparison to normal cells. By “amplification” is meantincreased numbers of DNA or RNA in a cell compared with normal cells.

[0178] The diseases that could be diagnosed by detection of nucleic acidin a sample preferably include central nervous system and metabolicdiseases. The test samples suitable for nucleic acid probing methods ofthe present invention include, for example, cells or nucleic acidextracts of cells, or biological fluids. The samples used in theabove-described methods will vary based on the assay format, thedetection method and the nature of the tissues, cells or extracts to beassayed. Methods for preparing nucleic acid extracts of cells are wellknown in the art and can be readily adapted in order to obtain a samplethat is compatible with the method utilized.

[0179] Alternatively, immunoassay kits can be provided which havecontainers container having antibodies specific for the MMP-protein andoptionally, containers with positive and negative controls and/orinstructions.

[0180] Kits may also be provided useful in the identification of MMPbinding partners such as natural ligands or modulators (agonists orantagonists). Substances useful for treatment of disorders or diseasespreferably show positive results in one or more in vitro assays for anactivity corresponding to treatment of the disease or disorder inquestion. Substances that modulate the activity of the polypeptidespreferably include, but are not limited to, antisense oligonucleotides,agonists and antagonists, and inhibitors of protein kinases.

[0181] Methods of Inducing Immune Response

[0182] Another aspect of the present invention is directed to methods ofinducing an immune response in a mammal against a polypeptide of theinvention by administering to the mammal an amount of the polypeptidesufficient to induce an immune response. The amount will be dependent onthe animal species, size of the animal, and the like but can bedetermined by those skilled in the art.

[0183] Methods of Identifying Ligands

[0184] The invention also provides assays to identify compounds thatbind MMP. One such assay comprises the steps of: (a) contacting acomposition comprising a MMP with a compound suspected of binding MMP;and (b) measuring binding between the compound and MMP. In onevariation, the composition comprises a cell expressing MMP on itssurface. In another variation, isolated MMP or cell membranes comprisingMMP are employed. The binding may be measured directly, e.g., by using alabeled compound, or may be measured indirectly. Following steps (a) and(b), compounds identified as binding MMP may be tested in other assaysincluding, but not limited to, in vivo models, to confirm or quantitatebinding to

[0185] Specific binding molecules, including natural ligands andsynthetic compounds, can be identified or developed using isolated orrecombinant MMP products, MMP variants, or preferably, cells expressingsuch products. Binding partners are useful for purifying MMP productsand detection or quantification of MMP products in fluid and tissuesamples using known immunological procedures. Binding molecules are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) biological activities of MMP, especially those activitiesinvolved in collagenase or proteinase activity.

[0186] The DNA and amino acid sequence information provided by thepresent invention also makes possible identification of binding partnercompounds with which an MMP polypeptide or polynucleotide will interact.Methods to identify binding partner compounds include solution assays,in vitro assays wherein MMP polypeptides are immobilized, and cell-basedassays. Identification of binding partner compounds of MMP polypeptidesprovides candidates for therapeutic or prophylactic intervention inpathologies associated with MMP normal and aberrant biological activity.

[0187] The invention includes several assay systems for identifying MMPbinding partners. In solution assays, methods of the invention comprisethe steps of (a) contacting a MMP polypeptide with one or more candidatebinding partner compounds and (b) identifying the compounds that bind tothe MMP polypeptide. Identification of the compounds that bind the MMPpolypeptide can be achieved by isolating the MMP polypeptide/bindingpartner complex, and separating the binding partner compound from theMMP polypeptide. An additional step of characterizing the physical,biological, and/or biochemical properties of the binding partnercompound is also comprehended in another embodiment of the invention,wherein compounds identified as binding MMP may be tested in otherassays including, but not limited to, in vivo models, to confirm orquantitate binding to MMP. In one aspect, the MMP polypeptide/bindingpartner complex is isolated using an antibody immunospecific for eitherthe MMP polypeptide or the candidate binding partner compound.

[0188] In still other embodiments, either the MMP polypeptide or thecandidate binding partner compound comprises a label or tag thatfacilitates its isolation, and methods of the invention to identifybinding partner compounds include a step of isolating the MMPpolypeptide/binding partner complex through interaction with the labelor tag. An exemplary tag of this type is a poly-histidine sequence,generally around six histidine residues, that permits isolation of acompound so labeled using nickel chelation. Other labels and tags, suchas the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known androutinely used in the art, are embraced by the invention.

[0189] In one variation of an in vitro assay, the invention provides amethod comprising the steps of (a) contacting an immobilized MMPpolypeptide with a candidate binding partner compound and (b) detectingbinding of the candidate compound to the MMP polypeptide. In analternative embodiment, the candidate binding partner compound isimmobilized and binding of MMP is detected. Immobilization isaccomplished using any of the methods well known in the art, includingcovalent bonding to a support, a bead, or a chromatographic resin, aswell as non-covalent, high affinity interactions such as antibodybinding, or use of streptavidin/biotin binding wherein the immobilizedcompound includes a biotin moiety. Detection of binding can beaccomplished (i) using a radioactive label on the compound that is notimmobilized, (ii) using of a fluorescent label on the non-immobilizedcompound, (iii) using an antibody immunospecific for the non-immobilizedcompound, (iv) using a label on the non-immobilized compound thatexcites a fluorescent support to which the immobilized compound isattached, as well as other techniques well known and routinely practicedin the art.

[0190] Another aspect of the present invention is directed to methods ofidentifying compounds that bind to either MMP or nucleic acid moleculesencoding MMP, comprising contacting MMP, or a nucleic acid moleculeencoding the same, with a compound, and determining whether the compoundbinds MMP or a nucleic acid molecule encoding the same. Binding can bedetermined by binding assays which are well known to the skilledartisan, including, but not limited to, gel-shift assays, Western blots,radiolabeled competition assay, phage-based expression cloning,co-fractionation by chromatography, co-precipitation, cross linking,interaction trap/two-hybrid analysis, southwestern analysis, ELISA, andthe like, which are described in, for example, Current Protocols inMolecular Biology, 1999, John Wiley & Sons, NY, which is incorporatedherein by reference in its entirety. The compounds to be screenedinclude (which may include compounds which are suspected to bind MMP, ora nucleic acid molecule encoding the same), but are not limited to,extracellular, intracellular, biologic or chemical origin. The methodsof the invention also embrace ligands, especially neuropeptides, thatare attached to a label, such as a radiolabel (e.g., ¹²⁵I, ³⁵S, ³²P,³³P, ³H), a fluorescence label, a chemiluminescent label, an enzymiclabel and an immunogenic label. Modulators falling within the scope ofthe invention include, but are not limited to, non-peptide moleculessuch as non-peptide mimetics, non-peptide allosteric effectors, andpeptides. The MMP polypeptide or polynucleotide employed in such a testmay either be free in solution, attached to a solid support, borne on acell surface or located intracellularly or associated with a portion ofa cell. One skilled in the art can, for example, measure the formationof complexes between MMP and the compound being tested. Alternatively,one skilled in the art can examine the diminution in complex formationbetween MMP and its substrate caused by the compound being tested.

[0191] In another embodiment of the invention, high throughput screeningfor compounds having suitable binding affinity to MMP is employed.Briefly, large numbers of different test compounds are synthesized on asolid substrate. The peptide test compounds are contacted with MMP andwashed. Bound MMP is then detected by methods well known in the art.Purified polypeptides of the invention can also be coated directly ontoplates for use in the aforementioned drug screening techniques. Inaddition, non-neutralizing antibodies can be used to capture the proteinand immobilize it on the solid support.

[0192] Generally, an expressed MMP can be used for HTS binding assays inconjunction with its defined ligand, in this case the correspondingneuropeptide that activates it. The identified peptide is labeled with asuitable radioisotope, including, but not limited to, ¹²⁵I, ³H, ³⁵S or³²P, by methods that are well known to those skilled in the art.Alternatively, the peptides may be labeled by well-known methods with asuitable fluorescent derivative (Baindur et al., Drug Dev. Res., 1994,33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160).Alternative methods include a scintillation proximity assay (SPA) or aFlashPlate format in which such separation is unnecessary (Nakayama,Cur. Opinion Drug Disc. Dev., 1998, 1, 85-91; Bosse et al., J.Biomolecular Screening, 1998, 3, 285-292.). Binding of fluorescentligands can be detected in various ways, including fluorescence energytransfer (FRET), direct spectrophotofluorometric analysis of boundligand, or fluorescence polarization (Rogers, Drug Discovery Today,1997, 2, 156-160; Hill, Cur. Opinion Drug Disc. Dev., 1998, 1, 92-97).

[0193] Other assays may be used to identify specific ligands of an MMP,including assays that identify ligands of the target protein throughmeasuring direct binding of test ligands to the target protein, as wellas assays that identify ligands of target proteins through affinityultrafiltration with ion spray mass spectroscopy/HPLC methods or otherphysical and analytical methods. Alternatively, such bindinginteractions are evaluated indirectly using the yeast two-hybrid systemdescribed in Fields et al., Nature, 340:245-246 (1989), and Fields etal., Trends in Genetics, 10:286-292 (1994), both of which areincorporated herein by reference. The two-hybrid system is a geneticassay for detecting interactions between two proteins or polypeptides.It can be used to identify proteins that bind to a known protein ofinterest, or to delineate domains or residues critical for aninteraction. Variations on this methodology have been developed to clonegenes that encode DNA binding proteins, to identify peptides that bindto a protein, and to screen for drugs. The two-hybrid system exploitsthe ability of a pair of interacting proteins to bring a transcriptionactivation domain into close proximity with a DNA binding domain thatbinds to an upstream activation sequence (UAS) of a reporter gene, andis generally performed in yeast. The assay requires the construction oftwo hybrid genes encoding (1) a DNA-binding domain that is fused to afirst protein and (2) an activation domain fused to a second protein.The DNA-binding domain targets the first hybrid protein to the UAS ofthe reporter gene; however, because most proteins lack an activationdomain, this DNA-binding hybrid protein does not activate transcriptionof the reporter gene. The second hybrid protein, which contains theactivation domain, cannot by itself activate expression of the reportergene because it does not bind the UAS. However, when both hybridproteins are present, the noncovalent interaction of the first andsecond proteins tethers the activation domain to the UAS, activatingtranscription of the reporter gene. For example, when the first proteinis a MMP gene product, or fragment thereof, that is known to interactwith another protein or nucleic acid, this assay can be used to detectagents that interfere with the binding interaction. Expression of thereporter gene is monitored as different test agents are added to thesystem. The presence of an inhibitory agent results in lack of areporter signal.

[0194] The yeast two-hybrid assay can also be used to identify proteinsthat bind to the gene product. In an assay to identify proteins thatbind to a MMP, or fragment thereof, a fusion polynucleotide encodingboth a MMP (or fragment) and a UAS binding domain (i.e., a firstprotein) may be used. In addition, a large number of hybrid genes eachencoding a different second protein fused to an activation domain areproduced and screened in the assay. Typically, the second protein isencoded by one or more members of a total cDNA or genomic DNA fusionlibrary, with each second protein-coding region being fused to theactivation domain. This system is applicable to a wide variety ofproteins, and it is not even necessary to know the identity or functionof the second binding protein. The system is highly sensitive and candetect interactions not revealed by other methods; even transientinteractions may trigger transcription to produce a stable mRNA that canbe repeatedly translated to yield the reporter protein.

[0195] Other assays may be used to search for agents that bind to thetarget protein. One such screening method to identify direct binding oftest ligands to a target protein is described in U.S. Pat. No.5,585,277, incorporated herein by reference. This method relies on theprinciple that proteins generally exist as a mixture of folded andunfolded states, and continually alternate between the two states. Whena test ligand binds to the folded form of a target protein (i.e., whenthe test ligand is a ligand of the target protein), the target proteinmolecule bound by the ligand remains in its folded state. Thus, thefolded target protein is present to a greater extent in the presence ofa test ligand which binds the target protein, than in the absence of aligand. Binding of the ligand to the target protein can be determined byany method that distinguishes between the folded and unfolded states ofthe target protein. The function of the target protein need not be knownin order for this assay to be performed. Virtually any agent can beassessed by this method as a test ligand, including, but not limited to,metals, polypeptides, proteins, lipids, polysaccharides, polynucleotidesand small organic molecules.

[0196] Another method for identifying ligands of a target protein isdescribed in Wieboldt et al., Anal. Chem., 69:1683-1691 (1997),incorporated herein by reference. This technique screens combinatoriallibraries of 20-30 agents at a time in solution phase for binding to thetarget protein. Agents that bind to the target protein are separatedfrom other library components by simple membrane washing. Thespecifically selected molecules that are retained on the filter aresubsequently liberated from the target protein and analyzed by HPLC andpneumatically assisted electrospray (ion spray) ionization massspectroscopy. This procedure selects library components with thegreatest affinity for the target protein, and is particularly useful forsmall molecule libraries.

[0197] Other embodiments of the invention comprise using competitivescreening assays in which neutralizing antibodies capable of binding apolypeptide of the invention specifically compete with a test compoundfor binding to the polypeptide. In this manner, the antibodies can beused to detect the presence of any peptide that shares one or moreantigenic determinants with MMP. Radiolabeled competitive bindingstudies are described in A. H. Lin et al. Antimicrobial Agents andChemotherapy, 1997, vol. 41, no. 10. pp. 2127-2131, the disclosure ofwhich is incorporated herein by reference in its entirety.

[0198] Identification of Modulating Agents

[0199] The invention also provides methods for identifying a modulatorof binding between a MMP and a MMP binding partner, comprising the stepsof: (a) contacting a MMP binding partner and a composition comprising aMMP in the presence and in the absence of a putative modulator compound;(b) detecting binding between the binding partner and the a MMP; and (c)identifying a putative modulator compound or a modulator compound inview of decreased or increased binding between the binding partner andthe MMP in the presence of the putative modulator, as compared tobinding in the absence of the putative modulator. Following steps (a)and (b), compounds identified as modulating binding between MMP and anMMP binding partner may be tested in other assays including, but notlimited to, in vivo models, to confirm or quantitate modulation ofbinding to MMP.

[0200] MMP binding partners that stimulate MMP activity are useful asagonists in disease states or conditions characterized by insufficientMMP activity. e.g., as a result of insufficient activity of a MMPligand). MMP binding partners that block ligand-mediated MMP signalingare useful as MMP antagonists to treat disease states or conditionscharacterized by excessive MMP signaling. In addition MMP modulators ingeneral, as well as MMP polynucleotides and polypeptides, are useful indiagnostic assays for such diseases or conditions.

[0201] In another aspect, the invention provides methods for treating adisease or abnormal condition by administering to a patient in need ofsuch treatment a substance that modulates the activity or expression ofa polypeptide having sequences selected from the group consisting of SEQID NO:4 to SEQ ID NO:6.

[0202] Agents that modulate (i.e., increase, decrease, or block) MMPactivity or expression may be identified by incubating a putativemodulator with a cell containing an MMP polypeptide or polynucleotideand determining the effect of the putative modulator on MMP activity orexpression. The selectivity of a compound that modulates the activity ofMMP can be evaluated by comparing its effects on MMP to its effect onother MMP compounds. Following identification of compounds that modulateMMP activity or expression, such compounds may be further tested inother assays including, but not limited to, in vivo models, in order toconfirm or quantitate their activity. Selective modulators may include,for example, antibodies and other proteins, peptides, or organicmolecules that specifically bind to an MMP polypeptide or a MMP-encodingnucleic acid. Modulators of MMP activity will be therapeutically usefulin treatment of diseases and physiological conditions in which normal oraberrant MMP activity is involved. MMP polynucleotides, polypeptides,and modulators may be used in the treatment of such diseases andconditions as metabolic diseases and disorders (e.g., type 2 diabetes,obesity, cardiovascular, dyslipidemias, adipogenesis, retinopathies,neuropathies, nephropathies etc.), proliferative diseases and cancers(e.g., different cancers such as breast, colon, lung, etc., tumorgrowth, tumor invasion, and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc.), hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.), CNS disorders (e.g., degenerative disorders such as Parkinson's,Alzheimer's, etc.), inflammatory conditions (e.g., Chron's disease,arthritis), diseases related to cell differentiation and homeostasis,cardiomyopathy, atherosclerosis, thromboembolic diseases, Sjögren'ssyndrome, renal failure, periodontal diseases, retinalneovascularization, wound healing, and neurodegenerative diseasesincluding, for example, Alzheimer's disease, multiple sclerosis,Parkinson's disease, and motoneuron disease, among others.

[0203] Methods of the invention to identify modulators includevariations on any of the methods described above to identify bindingpartner compounds, the variations including techniques wherein a bindingpartner compound has been identified and the binding assay is carriedout in the presence and absence of a candidate modulator. A modulator isidentified in those instances where binding between the MMP polypeptideand the binding partner compound changes in the presence of thecandidate modulator compared to binding in the absence of the candidatemodulator compound. A modulator that increases binding between the MMPpolypeptide and the binding partner compound is described as an enhanceror activator, and a modulator that decreases binding between the MMPpolypeptide and the binding partner compound is described as aninhibitor. Following identification of modulators, such compounds may befurther tested in other assays including, but not limited to, in vivomodels, in order to confirm or quantitate their activity as modulators.

[0204] The invention also comprehends high-throughput screening (HTS)assays to identify compounds that interact with or inhibit biologicalactivity (i.e., affect enzymatic activity, binding activity, etc.) of aMMP polypeptide. HTS assays permit screening of large numbers ofcompounds in an efficient manner. Cell-based HTS systems arecontemplated to investigate MMP-ligand interaction. HTS assays aredesigned to identify “hits” or “lead compounds” having the desiredproperty, from which modifications can be designed to improve thedesired property. Chemical modification of the “hit” or “lead compound”is often based on an identifiable structure/activity relationshipbetween the “hit” and the MMP polypeptide.

[0205] Another aspect of the present invention is directed to methods ofidentifying compounds which modulate (i.e., increase or decrease) anactivity of MMP comprising contacting MMP with a compound, anddetermining whether the compound modifies activity of MMP. The activityin the presence of the test compared is measured to the activity in theabsence of the test compound. Where the activity of the samplecontaining the test compound is higher than the activity in the samplelacking the test compound, the compound will have increased activity.Similarly, where the activity of the sample containing the test compoundis lower than the activity in the sample lacking the test compound, thecompound will have inhibited activity. Following the identification ofcompounds that modulate an activity of MMP, such compounds can befurther tested in other assays including, but not limited to, in vivomodels, in order to confirm or quantitate their activity.

[0206] The present invention is particularly useful for screeningcompounds by using MMP in any of a variety of drug screening techniques.The compounds to be screened include (which may include compounds whichare suspected to modulate MMP activity), but are not limited to,extracellular, intracellular, biologic or chemical origin. The NMPpolypeptide employed in such a test may be in any form, preferably, freein solution, attached to a solid support, borne on a cell surface orlocated intracellularly. One skilled in the art can, for example,measure the formation of complexes between MMP and the compound beingtested. Alternatively, one skilled in the art can examine the diminutionin complex formation between MMP and its substrate caused by thecompound being tested.

[0207] The activity of MMP polypeptides of the invention can bedetermined by, for example, examining the ability to bind or beactivated by appropriate ligands, such as low molecular weight steroidsand fatty acids. The activity of the MMPs can be assayed by, forexample, competition-binding assays and coactivator-interaction assays(see, e.g., Makishima et al, 1999, Science, 284, 1362-1365; Parks etal., Science, 284, 1365-1367). Alternatively, the activity of MMPpolypeptides can be assayed by examining their ability to cleave a knownMMP substrate.

[0208] The modulators of the invention exhibit a variety of chemicalstructures, which can be generally grouped into non-peptide mimetics ofnatural NMP ligands, peptide and non-peptide allosteric effectors ofMMPs, and peptides that may function as activators or inhibitors(competitive, uncompetitive and non-competitive) (e.g., antibodyproducts) of MMPs. The invention does not restrict the sources forsuitable modulators, which may be obtained from natural sources such asplant, animal or mineral extracts, or non-natural sources such as smallmolecule libraries, including the products of combinatorial chemicalapproaches to library construction, and peptide libraries.

[0209] Other assays can be used to examine enzymatic activity including,but not limited to, photometric, radiometric, HPLC, electrochemical, andthe like, which are described in, for example, Enzyme Assays: APractical Approach, eds. R. Eisenthal and M. J. Danson, 1992, OxfordUniversity Press, which is incorporated herein by reference in itsentirety.

[0210] The use of cDNAs encoding MMPs in drug discovery programs iswell-known; assays capable of testing thousands of unknown compounds perday in high-throughput screens (HTSs) are thoroughly documented. Theliterature is replete with examples of the use of radiolabeled ligandsin HoS binding assays for drug discovery (see Williams, MedicinalResearch Reviews, 1991, 11, 147-184; Sweetnam, et al., J. NaturalProducts, 1993, 56, 441-455 for review).

[0211] A variety of heterologous systems is available for functionalexpression of recombinant polypeptides that are well known to thoseskilled in the art. Such systems include bacteria (Strosberg, et al.,Trends in Pharmacological Sciences, 1992, 13, 95-98), yeast (Pausch,Trends in Biotechnology, 1997, 15, 487-494), several kinds of insectcells (Vanden Broeck, Int. Rev. Cytology, 1996, 164, 189-268), amphibiancells (Jayawickreme et al., Current Opinion in Biotechnology, 1997, 8,629-634) and several mammalian cell lines (CHO, HEK-293, COS, etc.; seeGerhardt, et al., Eur. J. Pharmacology, 1997, 334, 1-23). These examplesdo not preclude the use of other possible cell expression systems,including cell lines obtained from nematodes (PCT application WO98/37177).

[0212] In preferred embodiments of the invention, methods of screeningfor compounds that modulate MMP activity comprise contacting testcompounds with MMP and assaying for the presence of a complex betweenthe compound and MMP. In such assays, the ligand is typically labeled.After suitable incubation, free ligand is separated from that present inbound form, and the amount of free or uncomplexed label is a measure ofthe ability of the particular compound to bind to MMP.

[0213] The invention contemplates a multitude of assays to screen andidentify inhibitors of ligand binding to MMPs. In one example, the MMPis immobilized and interaction with a binding partner is assessed in thepresence and absence of a candidate modulator such as an inhibitorcompound. In another example, interaction between the MMP and itsbinding partner is assessed in a solution assay, both in the presenceand absence of a candidate inhibitor compound. In either assay, aninhibitor is identified as a compound that decreases binding between theMMP and its binding partner. Following the identification of compoundswhich inhibit ligand binding to MMP, such compounds may be furthertested in other assays including, but not limited to, in vivo models, inorder to confirm or quantitate their activity. Another contemplatedassay involves a variation of the dihybrid assay wherein an inhibitor ofprotein/protein interactions is identified by detection of a positivesignal in a transformed or transfected host cell, as described in PCTpublication number WO 95/20652, published Aug. 3, 1995.

[0214] Candidate modulators contemplated by the invention includecompounds selected from libraries of either potential activators orpotential inhibitors. There are a number of different libraries used forthe identification of small molecule modulators, including: (1) chemicallibraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides or organicmolecules. Chemical libraries consist of random chemical structures,some of which are analogs of known compounds or analogs of compoundsthat have been identified as “hits” or “leads” in other drug discoveryscreens, some of which are derived from natural products, and some ofwhich arise from non-directed synthetic organic chemistry. Naturalproduct libraries are collections of microorganisms, animals, plants, ormarine organisms which are used to create mixtures for screening by: (1)fermentation and extraction of broths from soil, plant or marinemicroorganisms or (2) extraction of plants or marine organisms. Naturalproduct libraries include polyketides, non-ribosomal peptides, andvariants (non-naturally occurring) thereof. For a review, see Science282:63-68 (1998). Combinatorial libraries are composed of large numbersof peptides, oligonucleotides, or organic compounds as a mixture. Theselibraries are relatively easy to prepare by traditional automatedsynthesis methods, PCR, cloning, or proprietary synthetic methods. Ofparticular interest are non-peptide combinatorial libraries. Still otherlibraries of interest include peptide, protein, peptidomimetic,multiparallel synthetic collection, recombinatorial, and polypeptidelibraries. For a review of combinatorial chemistry and libraries createdtherefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).Identification of modulators through use of the various librariesdescribed herein permits modification of the candidate “hit” (or “lead”)to optimize the capacity of the “hit” to modulate activity.

[0215] Still other candidate inhibitors contemplated by the inventioncan be designed and include soluble forms of binding partners, as wellas such binding partners as chimeric, or fusion, proteins. A “bindingpartner” as used herein broadly encompasses non-peptide modulators, aswell as such peptide modulators as neuropeptides other than naturalligands, antibodies, antibody fragments, and modified compoundscomprising antibody domains that are immunospecific for the expressionproduct of the identified MMP gene.

[0216] The polypeptides of the invention are employed as a research toolfor identification, characterization and purification of interacting,regulatory proteins. Appropriate labels are incorporated into thepolypeptides of the invention by various methods known in the art andthe polypeptides are used to capture interacting molecules. For example,molecules are incubated with the labeled polypeptides, washed to removeunbound polypeptides, and the polypeptide complex is quantified. Dataobtained using different concentrations of polypeptide are used tocalculate values for the number, affinity, and association ofpolypeptide with the protein complex.

[0217] Labeled polypeptides are also useful as reagents for thepurification of molecules with which the polypeptide interactsincluding, but not limited to, inhibitors. In one embodiment of affinitypurification, a polypeptide is covalently coupled to a chromatographycolumn. Cells and their membranes are extracted, and various cellularsubcomponents are passed over the column. Molecules bind to the columnby virtue of their affinity to the polypeptide. The polypeptide-complexis recovered from the column, dissociated and the recovered molecule issubjected to protein sequencing. This amino acid sequence is then usedto identify the captured molecule or to design degenerateoligonucleotides for cloning the corresponding gene from an appropriatecDNA library.

[0218] Alternatively, compounds may be identified which exhibit similarproperties to the ligand for the MMP of the invention, but which aresmaller and exhibit a longer half time than the endogenous ligand in ahuman or animal body. When an organic compound is designed, a moleculeaccording to the invention is used as a “lead” compound. The design ofmimetics to known pharmaceutically active compounds is a well-knownapproach in the development of pharmaceuticals based on such “lead”compounds. Mimetic design, synthesis and testing are generally used toavoid randomly screening a large number of molecules for a targetproperty. Furthermore, structural data deriving from the analysis of thededuced amino acid sequences encoded by the DNAs of the presentinvention are useful to design new drugs, more specific and thereforewith a higher pharmacological potency.

[0219] Comparison of the protein sequences of the present invention withthe sequences present in all the available databases showed homologywith the known MMPs. Accordingly, computer modeling can be used todevelop a putative tertiary structure of the proteins of the inventionbased on the available information of the conserved domains of thepolypeptides of the present invention. Thus, novel ligands based on thepredicted structure of MMP can be designed.

[0220] In a particular embodiment, the novel molecules identified by thescreening methods according to the invention are low molecular weightorganic molecules, in which case a composition or pharmaceuticalcomposition can be prepared thereof for oral intake, such as in tablets.The compositions, or pharmaceutical compositions, comprising the nucleicacid molecules, vectors, polypeptides, antibodies and compoundsidentified by the screening methods described herein, can be preparedfor any route of administration including, but not limited to, oral,intravenous, cutaneous, subcutaneous, nasal, intramuscular orintraperitoneal. The nature of the carrier or other ingredients willdepend on the specific route of administration and particular embodimentof the invention to be administered. Examples of techniques andprotocols that are useful in this context are, inter alia, found inRemington's Pharmaceutical Sciences, 16^(th) edition, Osol, A (ed.),1980, which is incorporated herein by reference in its entirety.

[0221] The dosage of these low molecular weight compounds will depend onthe disease state or condition to be treated and other clinical factorssuch as weight and condition of the human or animal and the route ofadministration of the compound. For treating human or animals, betweenapproximately 0.5 mg/kg of body weight to 500 mg/kg of body weight ofthe compound can be administered. Therapy is typically administered atlower dosages and is continued until the desired therapeutic outcome isobserved.

[0222] The present compounds and methods, including nucleic acidmolecules, polypeptides, antibodies, compounds identified by thescreening methods described herein, have a variety of pharmaceuticalapplications and may be used, for example, to treat or preventunregulated cellular growth, such as cancer cell and tumor growth. In aparticular embodiment, the present molecules are used in gene therapy.For a review of gene therapy procedures, see e.g. Anderson, Science,1992, 256, 808-813, which is incorporated herein by reference in itsentirety.

[0223] The present invention also encompasses a method of agonizing(stimulating) or antagonizing a MMP natural binding partner associatedactivity in a mammal comprising administering to said mammal an agonistor antagonist to one of the above disclosed polypeptides in an amountsufficient to effect said agonism or antagonism. One embodiment of thepresent invention, then, is a method of treating diseases in a mammalwith an agonist or antagonist of the protein of the present inventioncomprises administering the agonist or antagonist to a mammal in anamount sufficient to agonize or antagonize MMP-associated functions.

[0224] Exemplary diseases and conditions amenable to treatment based onthe present invention include, but are not limited to, metabolicdiseases and disorders (e.g., type 2 diabetes, obesity, cardiovascular,dyslipidemias, adipogenesis, retinopathies, neuropathies, nephropathiesetc.), proliferative diseases and cancers (e.g., different cancers suchas breast, colon, lung, etc., tumor growth, tumor invasion, andhyperproliferative disorders such as psoriasis, prostate hyperplasia,etc.), hormonal disorders (e.g., male/female hormonal replacement,polycystic ovarian syndrome, alopecia, etc.), CNS disorders (e.g.,degenerative disorders such as Parkinson's, Alzheimer's, etc.),inflammatory conditions (e.g., Chron's disease, arthritis), diseasesrelated to cell differentiation and homeostasis, cardiomyopathy,atherosclerosis, thromboembolic diseases, Sjbgren's syndrome, renalfailure, periodontal diseases, retinal neovascularization, woundhealing, and neurodegenerative diseases including, for example,Alzheimer's disease, multiple sclerosis, Parkinson's disease, andmotoneuron disease, among others.

[0225] Methods of determining the dosages of compounds to beadministered to a patient and modes of administering compounds to anorganism are disclosed in U.S. application Ser. No. 08/702,282, filedAug. 23, 1996 and International patent publication number WO 96/22976,published Aug. 1, 1996, both of which are incorporated herein byreference in their entirety, including any drawings, figures or tables.Those skilled in the art will appreciate that such descriptions areapplicable to the present invention and can be easily adapted to it.

[0226] The proper dosage depends on various factors such as the type ofdisease being treated, the particular composition being used and thesize and physiological condition of the patient. Therapeuticallyeffective doses for the compounds described herein can be estimatedinitially from cell culture and animal models. For example, a dose canbe formulated in animal models to achieve a circulating concentrationrange that initially takes into account the IC₅₀ as determined in cellculture assays. The animal model data can be used to more accuratelydetermine useful doses in humans.

[0227] Plasma half-life and bio-distribution of the drug and metabolitesin the plasma, tumors and major organs can also be determined tofacilitate the selection of drugs most appropriate to inhibit adisorder. Such measurements can be carried out. For example, HPLCanalysis can be performed on the plasma of animals treated with the drugand the location of radiolabeled compounds can be determined usingdetection methods such as X-ray, CAT scan and MRI. Compounds that showpotent inhibitory activity in the screening assays, but have poorpharmacokinetic characteristics, can be optimized by altering thechemical structure and retesting. In this regard, compounds displayinggood pharmacokinetic characteristics can be used as a model.

[0228] Toxicity studies can also be carried out by measuring the bloodcell composition. For example, toxicity studies can be carried out in asuitable animal model as follows: 1) the compound is administered tomice (an untreated control mouse should also be used); 2) blood samplesare periodically obtained via the tail vein from one mouse in eachtreatment group; and 3) the samples are analyzed for red and white bloodcell counts, blood cell composition and the percent of lymphocytesversus polymorphonuclear cells. A comparison of results for each dosingregime with the controls indicates if toxicity is present.

[0229] At the termination of each toxicity study, further studies can becarried out by sacrificing the animals (preferably, in accordance withthe American Veterinary Medical Association guidelines Report of theAmerican Veterinary Medical Assoc. Panel on Euthanasia, Journal ofAmerican Veterinary Medical Assoc., 202:229-249, 1993). Representativeanimals from each treatment group can then be examined by gross necropsyfor immediate evidence of metastasis, unusual illness or toxicity. Grossabnormalities in tissue are noted and tissues are examinedhistologically. Compounds causing a reduction in body weight or bloodcomponents are less preferred, as are compounds having an adverse effecton major organs. In general, the greater the adverse effect the lesspreferred the compound.

[0230] For the treatment of many diseases, the expected daily dose of ahydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably1 to 250 mg/day, and most preferably 1 to 50 mg/day. Drugs can bedelivered less frequently provided plasma levels of the active moietyare sufficient to maintain therapeutic effectiveness. Plasma levelsshould reflect the potency of the drug. Generally, the more potent thecompound the lower the plasma levels necessary to achieve efficacy.

[0231] As discussed above, it is well known that MMPs are expressed inmany different tissues and regions, including in the brain. MMP mRNAtranscripts may found in many other tissues, including, but not limitedto pancreas (and particularly pancreatic islet tissue), pituitary,skeletal muscle, adipose tissue, liver, and thyroid, and may be found inmany other tissues.

[0232] Sequences selected from the group consisting of SEQ ID NO:1 toSEQ ID NO:3 will, as detailed above, enable screening the endogenoushormones/ligands which activate, agonize, or antagonize MMP and forcompounds with potential utility in treating disorders including, butnot limited to, metabolic diseases and disorders (e.g., type 2 diabetes,obesity, cardiovascular, dyslipidemias, adipogenesis, retinopathies,neuropathies, nephropathies etc.), proliferative diseases and cancers(e.g., different cancers such as breast, colon, lung, etc., tumorgrowth, tumor invasion, and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc.), hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.), CNS disorders (e.g., degenerative disorders such as Parkinson's,Alzheimer's, etc.), inflammatory conditions (e.g., Chron's disease,arthritis), diseases related to cell differentiation and homeostasis,cardiomyopathy, atherosclerosis, thromboembolic diseases, Sjögren'ssyndrome, renal failure, periodontal diseases, retinalneovascularization, wound healing, and neurodegenerative diseasesincluding, for example, Alzheimer's disease, multiple sclerosis,Parkinson's disease, and motoneuron disease, among others.

[0233] The attached Sequence Listing contains the sequences of thepolynucleotides and polypeptides of the invention and is incorporatedherein by reference in its entirety.

[0234] Methods of Screening Human Subjects

[0235] Thus in yet another embodiment, the invention provides geneticscreening procedures that entail analyzing a person's genome—inparticular their alleles for the MMPs of the invention—to determinewhether the individual possesses a genetic characteristic found in otherindividuals that are considered to be afflicted with, or at risk for,developing a mental disorder or disease of the brain that is suspectedof having a hereditary component. For example, in one embodiment, theinvention provides a method for determining a potential for developing adisorder affecting the brain in a human subject comprising the steps ofanalyzing the coding sequence of one or more MMP genes from the humansubject; and determining development potential for the disorder in saidhuman subject from the analyzing step.

[0236] More particularly, the invention provides a method of screening ahuman subject to diagnose a disorder affecting the brain or geneticpredisposition therefor, comprising the steps of: (a) assaying nucleicacid of a human subject to determine a presence or an absence of amutation altering the amino acid sequence, expression, or biologicalactivity of at least one MMP that is expressed in the brain, wherein theMMP comprises an amino acid sequence selected from the group consistingof SEQ ID NO:1 to SEQ ID NO:3, or an allelic variant thereof, andwherein the nucleic acid corresponds to the gene encoding the MMP; and(b) diagnosing the disorder or predisposition from the presence orabsence of said mutation, wherein the presence of a mutation alteringthe amino acid sequence, expression, or biological activity of allele inthe nucleic acid correlates with an increased risk of developing thedisorder.

[0237] By “human subject” is meant any human being, human embryo, orhuman fetus. It will be apparent that methods of the present inventionwill be of particular interest to individuals that have themselves beendiagnosed with a disorder affecting the brain or have relatives thathave been diagnosed with a disorder affecting the brain.

[0238] By “screening for an increased risk” is meant determination ofwhether a genetic variation exists in the human subject that correlateswith a greater likelihood of developing a disorder affecting the brainthan exists for the human population as a whole, or for a relevantracial or ethnic human sub-population to which the individual belongs.Both positive and negative determinations (i.e., determinations that agenetic predisposition marker is present or is absent) are intended tofall within the scope of screening methods of the invention. Inpreferred embodiments, the presence of a mutation altering the sequenceor expression of at least one MMP allele in the nucleic acid iscorrelated with an increased risk of developing mental disorder, whereasthe absence of such a mutation is reported as a negative determination.

[0239] The “assaying” step of the invention may involve any techniquesavailable for analyzing nucleic acid to determine its characteristics,including but not limited to well-known techniques such as single-strandconformation polymorphism analysis (SSCP) [Orita et al., Proc Natl.Acad. Sci. USA, 86: 2766-2770 (1989)]; heteroduplex analysis [White etal., Genomics, 12: 301-306 (1992)]; denaturing gradient gelelectrophoresis analysis [Fischer et al., Proc. Natl. Acad. Sci. USA,80: 1579-1583 (1983); and Riesner et al., Electrophoresis, 10: 377-389(1989)]; DNA sequencing; RNase cleavage [Myers et al., Science, 230:1242-1246 (1985)]; chemical cleavage of mismatch techniques [Rowley etal., Genomics, 30: 574-582 (1995); and Roberts et al., Nucl. Acids Res.,25: 3377-3378 (1997)]; restriction fragment length polymorphismanalysis; single nucleotide primer extension analysis [Shumaker et al.,Hum. Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7:606-614 (1997)]; 5′ nuclease assays [Pease et al., Proc. Natl. Acad.Sci. USA, 91:5022-5026 (1994)]; DNA Microchip analysis [Ramsay, G.,Nature Biotechnology, 16: 40-48 (1999); and Chee et al., U.S. Pat. No.5,837,832]; and ligase chain reaction [Whiteley et al., U.S. Pat. No.5,521,065]. [See generally, Schafer and Hawkins, Nature Biotechnology,16: 33-39 (1998).] All of the foregoing documents are herebyincorporated by reference in their entirety.

[0240] Thus, in one preferred embodiment involving screening MMPsequences, for example, the assaying step comprises at least oneprocedure selected from the group consisting of: (a) determining anucleotide sequence of at least one codon of at least one MMP allele ofthe human subject; (b) performing a hybridization assay to determinewhether nucleic acid from the human subject has a nucleotide sequenceidentical to or different from one or more reference sequences; (c)performing a polynucleotide migration assay to determine whether nucleicacid from the human subject has a nucleotide sequence identical to ordifferent from one or more reference sequences; and (d) performing arestriction endonuclease digestion to determine whether nucleic acidfrom the human subject has a nucleotide sequence identical to ordifferent from one or more reference sequences.

[0241] In a highly preferred embodiment, the assaying involvessequencing of nucleic acid to determine nucleotide sequence thereof,using any available sequencing technique. [See, e.g., Sanger et al.,Proc. Natl. Acad. Sci. (USA), 74: 5463-5467 (1977) (dideoxy chaintermination method); Mirzabekov, TIBTECH, 12: 27-32 (1994) (sequencingby hybridization); Drmanac et al., Nature Biotechnology, 16: 54-58(1998); U.S. Pat. No. 5,202,231; and Science, 260: 1649-1652 (1993)(sequencing by hybridization); Kieleczawa et al., Science, 258:1787-1791 (1992) (sequencing by primer walking); (Douglas et al.,Biotechniques, 14: 824-828 (1993) (Direct sequencing of PCR products);and Akane et al., Biotechniques 16: 238-241 (1994); Maxam and Gilbert,Meth. Enzymol., 65: 499-560 (1977) (chemical termination sequencing),all incorporated herein by reference.] The analysis may entailsequencing of the entire MMP gene genomic DNA sequence, or portionsthereof; or sequencing of the entire MMP coding sequence or portionsthereof. In some circumstances, the analysis may involve a determinationof whether an individual possesses a particular allelic variant, inwhich case sequencing of only a small portion of nucleic acid—enough todetermine the sequence of a particular codon characterizing the allelicvariant—is sufficient. This approach is appropriate, for example, whenassaying to determine whether one family member inherited the sameallelic variant that has been previously characterized for anotherfamily member, or, more generally, whether a person's genome contains anallelic variant that has been previously characterized and correlatedwith a mental disorder having a heritable component.

[0242] In another highly preferred embodiment, the assaying stepcomprises performing a hybridization assay to determine whether nucleicacid from the human subject has a nucleotide sequence identical to ordifferent from one or more reference sequences. In a preferredembodiment, the hybridization involves a determination of whethernucleic acid derived from the human subject will hybridize with one ormore oligonucleotides, wherein the oligonucleotides have nucleotidesequences that correspond identically to a portion of the MMP genesequence taught herein, or that correspond identically except for onemismatch. The hybridization conditions are selected to differentiatebetween perfect sequence complementarity and imperfect matches differingby one or more bases. Such hybridization experiments thereby can providesingle nucleotide polymorphism sequence information about the nucleicacid from the human subject, by virtue of knowing the sequences of theoligonucleotides used in the experiments.

[0243] Several of the techniques outlined above involve an analysiswherein one performs a polynucleotide migration assay, e.g., on apolyacrylamide electrophoresis gel (or in a capillary electrophoresissystem), under denaturing or non-denaturing conditions. Nucleic acidderived from the human subject is subjected to gel electrophoresis,usually adjacent to (or co-loaded with) one or more reference nucleicacids, such as reference MMP encoding sequences having a coding sequenceidentical to all or a portion of SEQ ID NOS: 1 to 3 (or identical exceptfor one known polymorphism). The nucleic acid from the human subject andthe reference sequence(s) are subjected to similar chemical or enzymatictreatments and then electrophoresed under conditions whereby thepolynucleotides will show a differential migration pattern, unless theycontain identical sequences. [See generally Ausubel et al. (eds.),Current Protocols in Molecular Biology, New York: John Wiley & Sons,Inc. (1987-1999); and Sambrook et al., (eds.), Molecular Cloning, ALaboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1989), both incorporated herein by reference in theirentirety.]

[0244] In the context of assaying, the term “nucleic acid of a humansubject” is intended to include nucleic acid obtained directly from thehuman subject (e.g., DNA or RNA obtained from a biological sample suchas a blood, tissue, or other cell or fluid sample); and also nucleicacid derived from nucleic acid obtained directly from the human subject.By way of non-limiting examples, well known procedures exist forcreating cDNA that is complementary to RNA derived from a biologicalsample from a human subject, and for amplifying (e.g., via polymerasechain reaction (PCR)) DNA or RNA derived from a biological sampleobtained from a human subject. Any such derived polynucleotide whichretains relevant nucleotide sequence information of the human subject'sown DNA/RNA is intended to fall within the definition of “nucleic acidof a human subject” for the purposes of the present invention.

[0245] In the context of assaying, the term “mutation” includesaddition, deletion, and/or substitution of one or more nucleotides inthe MMP gene sequence (e.g., as compared to the MMP-encoding sequencesset forth of SEQ ID NO:1 to SEQ ID NO:3, and other polymorphisms thatoccur in introns (where introns exist) and that are identifiable viasequencing, restriction fragment length polymorphism, or othertechniques. The various activity examples provided herein permitdetermination of whether a mutation modulates activity of the relevantMMP in the presence or absence of various test substances.

[0246] In a related embodiment, the invention provides methods ofscreening a person's genotype with respect to the MMP of the invention,and correlating such genotypes with diagnoses for disease or withpredisposition for disease (for genetic counseling). For example, theinvention provides a method of screening for an MMP hereditary mentaldisorder genotype in a human patient, comprising the steps of: (a)providing a biological sample comprising nucleic acid from the patient,the nucleic acid including sequences corresponding to said patient's MMPalleles; (b) analyzing the nucleic acid for the presence of a mutationor mutations; (c) determining a MMP genotype from the analyzing step;and (d) correlating the presence of a mutation in an MMP allele with ahereditary mental disorder genotype. In a preferred embodiment, thebiological sample is a cell sample containing human cells that containgenomic DNA of the human subject. The analyzing can be performedanalogously to the assaying described in preceding paragraphs. Forexample, the analyzing comprises sequencing a portion of the nucleicacid (e.g., DNA or RNA), the portion comprising at least one codon ofthe MMP alleles.

[0247] Although more time consuming and expensive than methods involvingnucleic acid analysis, the invention also may be practiced by assayingone or more proteins of a human subject to determine the presence orabsence of an amino acid sequence variation in MMP protein from thehuman subject. Such protein analyses may be performed, e.g., byfragmenting MMP protein via chemical or enzymatic methods and sequencingthe resultant peptides; or by Western analyses using an antibody havingspecificity for a particular allelic variant of the MMP.

[0248] The invention also provides materials that are useful forperforming methods of the invention. For example, the present inventionprovides oligonucleotides useful as probes in the many analyzingtechniques described above. In general, such oligonucleotide probescomprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides that have asequence that is identical, or exactly complementary, to a portion of ahuman MMP gene sequence taught herein (or allelic variant thereof), orthat is identical or exactly complementary except for one nucleotidesubstitution. In a preferred embodiment, the oligonucleotides have asequence that corresponds in the foregoing manner to a human MMP codingsequence taught herein, and in particular, the coding sequences setforth in SEQ ID NO:1 to SEQ ID NO:3. In one variation, anoligonucleotide probe of the invention is purified and isolated. Inanother variation, the oligonucleotide probe is labeled, e.g., with aradioisotope, chromophore, or fluorophore. In yet another variation, theprobe is covalently attached to a solid support. [See generally Ausubelet al. and Sambrook et al., supra.]

[0249] In a related embodiment, the invention provides kits comprisingreagents that are useful for practicing methods of the invention. Forexample, the invention provides a kit for screening a human subject todiagnose a mental disorder or a genetic predisposition therefor,comprising, in association: (a) an oligonucleotide useful as a probe foridentifying polymorphisms in a human MMP gene, the oligonucleotidecomprising 6-50 nucleotides that have a sequence that is identical orexactly complementary to a portion of a human MMP gene sequence or MMPcoding sequence, except for one sequence difference selected from thegroup consisting of a nucleotide addition, a nucleotide deletion, ornucleotide substitution; and (b) a media packaged with theoligonucleotide containing information identifying polymorphismsidentifiable with the probe that correlate with mental disorder or agenetic predisposition therefor. Exemplary information-containing mediainclude printed paper package inserts or packaging labels; and magneticand optical storage media that are readable by computers or machinesused by practitioners who perform genetic screening and counselingservices. The practitioner uses the information provided in the media tocorrelate the results of the analysis with the oligonucleotide with adiagnosis. In a preferred variation, the oligonucleotide is labeled.

[0250] In still another embodiment, the invention provides methods ofidentifying those allelic variants of MMP of the invention thatcorrelate with mental disorders. For example, the invention provides amethod of identifying an MMP allelic variant that correlates with amental disorder, comprising steps of: (a) providing a biological samplecomprising nucleic acid from a human patient diagnosed with a mentaldisorder, or from the patient's genetic progenitors or progeny; (b)analyzing the nucleic acid for the presence of a mutation or mutationsin at least one MMP that is expressed in the brain, wherein the at leastone MMP comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:3 or an allelic variant thereof,and wherein the nucleic acid includes sequence corresponding to the geneor genes encoding the at least one MMP; (c) determining a genotype forthe patient for the at least one MMP from said analyzing step; and (d)identifying an allelic variant that correlates with the mental disorderfrom the determining step. To expedite this process, it may be desirableto perform linkage studies in the patients (and possibly their families)to correlate chromosomal markers with disease states. The chromosomallocalization data provided herein facilitates identifying an involvedMMP with a chromosomal marker.

[0251] The foregoing method can be performed to correlate the MMP of theinvention to a number of disorders having hereditary components that arecausative or that predispose persons to the disorder. For example, inone preferred variation, the disorder is a mental disorder.

[0252] Also contemplated as part of the invention are polynucleotidesthat comprise the allelic variant sequences identified by such methods,and polypeptides encoded by the allelic variant sequences, andoligonucleotide and oligopeptide fragments thereof that embody themutations that have been identified. Such materials are useful in invitro cell-free and cell-based assays for identifying lead compounds andtherapeutics for treatment of the disorders. For example, the variantsare used in activity assays, binding assays, and assays to screen foractivity modulators described herein. In one preferred embodiment, theinvention provides a purified and isolated polynucleotide comprising anucleotide sequence encoding a MMP allelic variant identified accordingto the methods described above; and an oligonucleotide that comprisesthe sequences that differentiate the allelic variant from the MMPsequences set forth in SEQ ID NO:1 to SEQ ID NO:3. The invention alsoprovides a vector comprising the polynucleotide (preferably anexpression vector); and a host cell transformed or transfected with thepolynucleotide or vector. The invention also provides an isolated cellline that is expressing the allelic variant NMP polypeptide; purifiedcell membranes from such cells; purified polypeptide; and syntheticpeptides that embody the allelic variation amino acid sequence. In oneparticular embodiment, the invention provides a purified polynucleotidecomprising a nucleotide sequence encoding an MMP protein of a human thatis affected with a mental disorder; wherein said polynucleotidehybridizes to the complement of a sequence selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:3 under the followinghybridization conditions: (a) hybridization for 16 hours at 42° C. in ahybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10%dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in awash solution comprising 0.1×SSC and 1% SDS; and wherein thepolynucleotide encodes an MM amino acid sequence that differs from asequence selected from the group consisting of SEQ ID NO:4 to SEQ IDNO:6, by at least one residue.

[0253] An exemplary assay for using the allelic variants is a method foridentifying a modulator of MMP biological activity, comprising the stepsof: (a) contacting a cell expressing the allelic variant in the presenceand in the absence of a putative modulator compound; (b) measuring MMPbiological activity in the cell; and (c) identifying a putativemodulator compound in view of decreased or increased MMP biologicalactivity in the presence versus absence of the putative modulator.

[0254] Additional features of the invention will be apparent from thefollowing Examples. Examples 1 is actual while the remaining Examplesare prophetic. Additional features and variations of the invention willbe apparent to those skilled in the art from the entirety of thisapplication, including the detailed description, and all such featuresare intended as aspects of the invention. Likewise, features of theinvention described herein can be re-combined into additionalembodiments that also are intended as aspects of the invention,irrespective of whether the combination of features is specificallymentioned above as an aspect or embodiment of the invention. Also, onlysuch limitations which are described herein as critical to the inventionshould be viewed as such; variations of the invention lackinglimitations which have not been described herein as critical areintended as aspects of the invention.

EXAMPLE 1 Identification of MMP

[0255] A. Database Search

[0256] The Incyte LifeSeq databases LGTemplatesAUG1999,LGTemplatesOCT1999, and LGTemplatesDEC1999, and the Celera databaseReleases 1.04-1.05 were searched using BLAST and nucleotide/proteinsequence from known MMPs. A collection of query amino acid sequencesderived from MMPs was used to search the DNA sequence databases usingTBLASTN and alignments with an E-value lower than 10⁻⁵ were collectedfrom each BLAST search. The new sequences found were then BLAST searchedagainst proprietary databases in order to eliminate known MMPs andidentify novel MMPs.

[0257] Briefly, the BLAST algorithm (Basic Local Alignment Search Tool)is suitable for determining sequence similarity (Altschul et al., J.Mol. Biol., 1990, 215, 403-410, which is incorporated herein byreference in its entirety). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence that either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind HSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extension for the word hits in each direction are haltedwhen: 1) the cumulative alignment score falls off by the quantity X fromits maximum achieved value; 2) the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or 3) the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad.Sci. USA, 1992, 89, 10915-10919, which is incorporated herein byreference in its entirety) alignments (B) of 50, expectation (E) of 10,M=5, N=4, and a comparison of both strands.

[0258] The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA,1993, 90, 5873-5787, which is incorporated herein by reference in itsentirety) and Gapped BLAST perform a statistical analysis of thesimilarity between two sequences. One measure of similarity provided bythe BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a MMP gene or cDNA if the smallestsum probability in comparison of the test nucleic acid to an MMP nucleicacid is less than about 1 to about 10⁻⁵.

[0259] The following Table 5 contains the sequences of thepolynucleotides and polypeptides of the invention. The coding regionswithin the polynucleotide sequences are identified by underlining. TABLE5 The following DNA sequence MMPU1 <SEQ ID NO. 1> was identified in H.sapiens.GCTTCAGCTGAAGAAAGAGAGGAATGAAGCGCCTTCTGCTTCTGTGTTTGTTCTTTATAACATTTTCTTCTGCATTTCCCTTAGTCCGGATGACGGAAAATGAAGAAAATATGCAACTGGCTCAGGCATATCTCAACCAGTTCTACTCTCTTGAAATAGAAGCGAATCATCTTGTTCAAAGCAAGAATAGGAGTCTCATAGATGACAAAATTCGGGAAATGCAAGCATTTTTTGGATTGACAGTGACTGGAAAACTGGACTCAAACACCCTTGAGATCATGAAGACACCCAGGTGTGGGGTGCCTGATGTGGGCCAGTATGGCTACACCCTCCCTGGGTGGAGAAAATACAACCTCACCTACAGAATAATAAACTATACTCCGGATATGGGACGAGCTGCTGTGGATGAGGCTATCCAAGAAGGTTTAGAAGTGTGGAGCAAAGTCACTCCACTAAAATTCACCAAGATTTCAAAGGGGATTGCAGACATCATGATTGCCTTTAGGACTCGAGTCCATGGTCCGTGTCCTCGCTATTTTGATGGTCCCTTCGGAGTGCTTGGCCATGCCTTTCCTCCTGGTCCGGCTCTGGGTGGTGACACTCATTTTGATGAGGATGAAAACTGGACCAAGGATGGAGCAGGATTCAACTTGTTTCTTGTGGCTGCTCATGAATTTGGTCATGCACTGGGGCTCTCTCACTCCAATGATCAAACAGCCTTGATGTTCCCAAATTATGTCTCCCTGGATCCCAGAAAATACCCACTTTCTCAGGATGATATCAATGGAATCCAGTCCATCTATGGAGGTCTGCCTAAGGTACCTGCTAAGCCAAAGGAACCCACTATACCCCATGCCTGTGACCCTGACTTGACTTTTGACGCTATCACAACTTTCCGCAGAGAAGTAATGTTCTTTAAAGGCAGGCACCTATGGAGGATCTATTATGATATCACGGATGTTGAGTTTGAATTAATTGCTTCATTCTGGCCATCTCTGCCAGCTGATCTGCAAGCTGCATACGAGAACCCCAGAGATAAGATTCTGGTTTTTAAAGATGAAAACTTCTGGATGATCAGAGGATATGCTGTCTTGCCAGATTATCCCAAATCCATCCATACATTAGGTTTTCCAGGACGTGTGAAGAAAATAGATGCAGCCGTCTGTGATAAGACCACAAGAAAAACCTACTTCTTTGTGGGCATTTGGTGCTGGAGGTTTGATGAAATGACCCAAACCATGGACAAAGGATTCCCGCAGAGAGTGGTAAAACACTTTCCTGGAATCAGTATCCGTGTTGATGCTGCTTTCCAGTACAAAGGATTCTTCTTTTTCAGCCGTGGATCAAAGCAATTTGAATACAACATTAAGACAAAGAATATTACCCGAATCATGAGAACTAATACTTGGTTTCAATCCAAAGAACCAAAGAACTCCTCATTTGGTTTTGATATCAACAAGGAAAAAGCACATTCAGGAGGCATAAAGATATTGTATCATAAGAGTTTAAGCTTGTTTATTTTTGGTATTGTTCATTTGCTGAAAAACACTTCTATTTATCAATAAATTCATAGACCTAAAATAAACCTCAACAGGTCTTTTAATATAAATTCTGCTTCAAAATAGAATAAAACCATTCTTTAACAACAAGTTGCTGGTCCTAGTTCTAAATATCCAAATTCAATGGCCATTTTGAGCTGCCTGATTCTTTTAATAGGAAGTTATTATGTAGAAACAAAAATCTCTGACTGTACTTTAAGCCTATTTCATGCTTTGTGGACTTGGAGAAGACATGTCTTATAACTGAATACTGAAACATTTATTAAACCAATCTTTAGCATTCTAAThe following amino acid sequence <SEQ ID NO. 4> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 1:MKRLLLLCLFFITFSSAFPLVRMTENEENMOLAOAYLNOFYSLEIEGNHLVQSKNRSLIDDKIREMQAFFGLTVTGKLDSNTLEIMKTPRCGVPDVGQYGYTLPGWRKYNLTYRIINYTPDMARAAVDEAIQEGLEVWSKVTPLKFTKISKGIADIMIAFRTRVHGRCPRYFDGPLGVLGHAFPPGPGLGGDTHFDEDENWTKDGAGFNLFLVAAHEFGHALGLSHSNDQTALMFPNYVSLDPRKYPLSQDDINGIQSIYGGLPKVPAKPKEPTIPHACDPDLTFDAITTFRREVMFFKGRHLWRIYYDITDVEFELIASFWPSLPADLQAAYENPRDKILVFKDENFWMIRGYAVLPDYPKSIHTLGFPGRVKKIDAAVCDKTTRKTYFFVGIWCWRFDEMTQTMDKGFPQRVVKHFPGISIRVDAAFQYKGFFFFSRGSKQFEYNIKTKNITRIMRTNTWFQCKEPKNSSFGFDINKEKAHSGGIKILYHKSLSLFIFGIVHLLKNTSIYQ The following DNA sequence MMPU9 <SEQ ID NO. 2> wasidentified in H. sapiens:GACAAATGAGGGTTTGGCATGCAGCTCGTCATCTTAAGAGTTACTATCTTCTTGCCCTGGTGTTTCGCCGTTCCAGTGCCCCCTGCTGCAGACCATAAAGGATGGGACTTTGTTGAGGGCTATTTCCATCAATTTTTCCTGACCAAGAAGGAGTCGCCACTCCTTACCCAGGAGACACAAACACAGCTCCTGCAACAATTCCATCGGAATGGGACAGACCTACTTGACATGCAGATGCATGCTCTGCTACACCAGCCCCACTGTGGGGTGCCTGATGGGTCCGACACCTCCATCTCGCCAGGAAGATGCAAGTGGAATAAGCACACTCTAACTTACAGGATTATCAATTACCCACATGATATGAAGCCATCCGCAGTGAAAGACAGTATATATAATGCAGTTTCCATCTGGAGCAATGTGACCCCTTTGATATTCCAGCAAGTGCAGAATGGAGATGCACACATCAAGGTTTCTTTCTGGCAGTGGGCCCATGAAGATGGTTGGCCCTTTGATGGGCCAGGTGGTATCTTAGGCCATGCCTTTTTACCAAATTCTGGAAATCCTGGAGTTGTCCATTTTGACAAGAATGAACACTGGTCAGCTTCAGACACTGGATATAATCTGTTCCTGGTTGCAACTCATGAGATTGGGCATTCTTTGGGCCTGCAGCACTCTGGGAATCAGAGCTCCATAATGTACCCCACTTACTGGTATCACGACCCTAGAACCTTCCAGCTCAGTGCCGATGATATCCAAAGGATCCAGCATTTGTATGGAGAAAAATGTTCATCTGACATACCTTAATGTTAGCACAGAGGACTTATTCAACCTGTCCTTTCAGGGAGTTTATTGGAGGATCAAAGAACTGAAAGCACTAGAGCAGCCTTGGGGACTGCTAGGATGAAGCCCTAAAGAATGCAACCTAGTCAGGTTAGCTGAACCGACACTCAAAACGCTACTGAGTCACAATAAAGATTGTTTTAAAGAGT Thefollowing amino acid sequence <SEQ ID NO. 5> is the predicted amino acidsequence derived from the DNA sequence of SEQ ID NO. 2:MQLVILRVTIFLPWCFAVPVPPAADHKGWDFVEGYFHQFFLTKKESPLLTQETQTQLLQQFHRNGTDLLDMQMHALLHQPHCGVPDGSDTSISPGRCKWNKHTLTYRIINYPHDMKPSAVKDSIYNAVSIWSNVTPLTFQQVQNGDADIKVSFWQWAHEDGWPFDGPGGILGHAFLPNSGNPGVVHFDKNEHWSASDTGYNLFLVATHEIGHSLGLQHSGNQSSIMYPTYWYHDPRTFQLSADDIQRIQHLYGEKCSSD The following DNAsequence MMPU1O <SEQ ID NO. 3> was identified in H. sapiens:GCTCCCCGAGCCGGGCTGCACCGGAGGCGGCGAGATCGTCCCGCGCGTCGGCCTCCTGCTGCGCGCCCTGCAGCTGCTACTGTGGGGCCACCTGGACGCCCAGCCCGCGGAGCGCGGAGGCCAGGAGCTGCGCAAGGAGGCGGAGGCATTCCTAGAGAAGTACGGATACCTCAATGAACAGGTCCCCAAAGCTCCCACCTCCACTCGATTCAGCGATGCCATCAGAGCGTTTCAGTGGGTGTCCCAGCTACCTGTCAGCGGCGTGTTGGACCGCGCCACCCTGCGCCAGATGACTCGTCCCCGCTGCGGGGTTACAGATACCAACAGTTATGCGGCCTGGGCTGAGAGGATCAGTGACTTGTTTGCTAGACACCGGACCAAAATGAGGCGTAAGAAACGCTTTGCAAAGCAAGGTAACAAATGGTACAAGCAGCACCTCTCCTACCGCCTGGTCAACTGCCCTGAGCATCTCCGGAGCCGGCAGTTCGGGGCGCCGTGCGCGCCGCCTTCCAGTTGTGGAGCAACGTCTCAGCGCTGGAGTTCTGGGAGGCCCCAGCCACAGGCCCCGCTGACATCCGGCTCACCTTCTTCCAAGGGGACCACAACGATGGGCTGGGCAATGCCTTTGATGGCCCAGGGGGCGCCCTGCCGCACGCCTTTCCTGCCCCGCCGCGGCGAAGCGCACTTCGACCAAGATGAGCGCTGGTCCCTGAGCCGCCGCCGCGGGCGCAACCTGTTCGTCGTGCTGGCGCACGAGATCGCTCACACGCTTGGCCTCACCCACTCGCCCGCGCCGCGCGCGCTCATGCCGCCCTACTACAAGAGGCTGGGCCGCGACGCGCTGCTCAGCTGGGACGACGTGCTGGCCGTGCAGAGCCTGTATGGCAAGCCCCTAGGGGGCTCAGTCGCCGTCCAGCTCCCAGGAAAGCTGTTCACTGACTTTGAGACCTGGGACTCCTACAGCCCCCAAGGAAGGCGCCCTGAAACGCAGGGCCCTAAATACTGCCACTCTTCCTTCGATGCCATCACTGTAGACAGGCAACAGCAACTGTACATTTTTAAAGGGAGCCATTTCTGCGAGGTGGCAGCTGATGGCAACGTCTCAGAGCCCCGTCCACTGCAGGAAAGATGGGTCGGGCTGCCCCCCAACATTGAGGCTGCGGCAGTGTCATTGAATGATGGAGATTTCTACTTCTTCAAAGGGGGTCGATGCTGGAGGTTCCGGGGCCCCAAGCCAGTGTGGGGTCTCCCACAGCTGTGCCGGGCAGGGGGCCTGCCCCGCCATCCTGACGCCGCCCTCTTCTTCCCTCCTCTGCGCCGCCTCATCCTCTTCAAGCGTGCCCGCTACTACGTGCTGGCCCGAGCGGGACTGCAAGTGGAGCCCTACTACCCCCGAAGTCTGCAGGACTGGGGAGCCATCCCTGAGGAGCTCAGCGGCGCCCTGCCGAGGCCCGATGCCTCCATCATCTTCTTCCGAGATGACCGCTACTGGCGCCTCGACCAGGCCAAACTGCAGGCAACCACCTCGGGCCGCTGGGCCACCGACCTGCCCTGGATGGGCTGCTGGCATGCCAACTCGGGGAGCGCCCTGTTCTGA The following amino acid sequence<SEQ ID NO. 6> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 3:MVARVGLLLRALOLLLWGHLDAQPAERGGQELRKEAEAFLEKYGYLNEQVPKAPTSTRFSDAIRAFQWVSQLPVSGVLDRATLRQMTRPRCGVTDTNSYAAWAERISDLFARHRTKMRRKKRFAKQGNKWYKQHLSYRLVNWPEHLRSRQFGAPCAPPSSCGATSQRWSSGRPQPQAPLTSGSPSSKGTTTMGWAMPLMAQGAPWRTPFLPRRGEAHFDQDERWSLSRRRGRNLFVVLAHEIGHTLGLTHSPAPRALMAPYYKRLGRDALLSWDDVLAVQSLYGKPLGGSVAVOLPGKLFTDFETWDSYSPQGRRPETQGPKYCHSSFDAITVDRQQQLYIFKGSHFWEVAADGNVSEPRPLQERWVGLPPNIEAAAVSLNDGDFYFFKGGRCWRFRGPKPVWGLPQLCRAGGLPRHPDAALFFPPLRRLILFKGARYYVLARGGLQVEPYYPRSLQDWGGIPEEVSGALPRPDGSIIFFRDDRYWRLDQAKLQATTSGRWATELPWNGCWHANSGSALF The following DNA sequence <SEQ ID NO. 7> wasidentified in H. sapiens:ggcacgagcatgcagctcgtcatcttaagagttactatcttcttgccctggtgtttcgccgttccagtgccccctgctgcagaccataaaggatgggactttgttgagggctatttccatcaatttttcctgaccgagaaggagtcgccactccttacccaggagacacaaacacagctcctgcaacaattccatcggaatgggacagacctacttgacatgcagatgcatgctctgctacaccagccccactgtggggtgcctgatgggtccgacacctccatctcgccaggaagatgcaagtggaataagcacactctaacttacaggattatcaattacccacatgatatgaagccatccgcagtgaaagacagtatatataatgcagtttccatctggagcaatgtgacccctttgatattccagcaagtgcagaatggagatgcagacatcaaggtttctttctggcagtgggcccatgaagatggttggccctttgatgggceaggtggtatcttaggccatgcctttttaccaaattctggaaatcctggagttgtccattttgacaagaatgaacactggtcagcttcagacactggatataatctgttcctggttgcaactcatgagattgggcattctttgggcctgcagcactctgggaatcagagctccataatgtaccccacttactggtatcacgaccctagaaccttccagctcagtgccgatgatatccaaaggatccagcatttgtatggagaaaaatgttcatctgacataccttaatgttagcacagaggacttattcaacctgtctttcagggagtttattggaggatcaaagaactgaaagcactagagcagccttggggactgctaggatgaagccctaaagaatgcaacctagtcaggttagctgaaccgacactcaaaacgctactgagtcacaataaagattgttttaaagagtaaaaaaaaaaaaaaaaaaaa The following amino acid sequence <SEQ ID NO. 8> is the amino acidsequence derived from the DNA sequence of SEQ ID NO. 7:MVRVTWCAVVAADHKGWDVGYHTKSTTTHRNGTDDMMHAHHCGVDGSDTSSGRCKWNKHTTYRNYHDMKSAVKDSYNAVSWSNVTVNGDADKVSWWAHDGWDGGGGHANSGNGVVHDKNHWSASDTGYNVATHGHSGHSGNSSNYTYWYHDRTSADDRHYGKCSSD

EXAMPLE 2 Cloning of MMP cDNA

[0260] cDNAs may be sequenced directly using an AB1377 or ABI373Afluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division,PE/ABD, Foster City, Calif.) and the ABI PRISM Ready Dye-DeoxyTerminator kit with Taq FS polymerase. Each ABI cycle sequencingreaction contains about 0.5 μg of plasmid DNA. Cycle-sequencing isperformed using an initial denaturation at 98° C. for 1 min, followed by50 cycles: 98° C. for 30 sec, annealing at 50° C. for 30 sec, andextension at 60° C. for 4 min. Temperature cycles and times arecontrolled by a Perkin-Elmer 9600 thermocycler. Extension products arepurified using Centriflex gel filtration (Advanced Genetic TechnologiesCorp., Gaithersburg, Md.). Each reaction product is loaded by pipetteonto the column, which is then centrifuged in a swinging bucketcentrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4min at room temperature. Column-purified samples are dried under vacuumfor about 40 min and then dissolved in 5 μl of a DNA loading solution(83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran). Thesamples are then heated to 90° C. for three minutes and loaded into thegel sample wells for sequence analysis by the ABI377 sequencer. Sequenceanalysis is performed by importing ABI373A files into the Sequencherprogram (Gene Codes, Ann Arbor, Mich.). Generally, sequence reads of 700bp are obtained. Potential sequencing errors are minimized by obtainingsequence information from both DNA strands and by re-sequencingdifficult areas using primers at different locations until allsequencing ambiguities are removed.

[0261] To isolate a cDNA clone encoding full length MMP, a DNA fragmentcorresponding to a nucleotide sequence of SEQ ID NOS:1-3, or a portionthereof, can be used as a probe for hybridization screening of a phagecDNA library. The DNA fragment is amplified by the polymerase chainreaction (PCR) method. The PCR reaction mixture of 50 ml containspolymerase mixture (0.2 mM dNTPs, 1×PCR Buffer and 0.75 ml Expand HighFidelity Polymerase (Roche Biochemicals)), 1 μg of 3206491 plasmid, 50pmoles of forward primer and 50 pmoles of reverse primer. The primersare preferably 10 to 25 nucleotides in length and are determined byprocedures well known to those skilled in the art. Amplification isperformed in an Applied Biosystems PE2400 thermocycler, using thefollowing program: 95° C. for 15 seconds, 52° C. for 30 seconds and 72°C. for 90 seconds; repeated for 25 cycles. The amplified product isseparated from the plasmid by agarose gel electrophoresis, and purifiedby Qiaquick™ gel extraction kit (Qiagen).

[0262] A lambda phage library containing cDNAs cloned into lambda ZAPIIphage-vector is plated with E. coli XL-1 blue host, on 15 cm LB-agarplates at a density of 50,000 pfu per plate, and grown overnight at 37°C.; (plated as described by Sambrook et al., supra). Phage plaques aretransferred to nylon membranes (Amersham Hybond N.J.), denatured for 2minutes in denaturation solution (0.5 M NaOH, 1.5 M NaCl), renatured for5 minutes in renaturation solution (1 M Tris pH 7.5, 1.5 M NaCI), andwashed briefly in 2×SSC (20×SSC: 3 M NaCl, 0.3 M Na-citrate). Filtermembranes are dried and incubated at 80° C. for 120 minutes tocross-link the phage DNA to the membranes.

[0263] The membranes are hybridized with a DNA probe prepared asdescribed above. A DNA fragment (25 ng) is labeled with α-³²P-dCTP (NEN)using Rediprime™ random priming (Amersham Pharmacia Biotech), accordingto the manufacturer's instructions. Labeled DNA is separated fromunincorporated nucleotides by S200 spin columns (Amersham PharmaciaBiotech), denatured at 95° C. for 5 minutes and kept on ice. TheDNA-containing membranes (above) are pre-hybridized in 50 ml ExpressHyb™(Clontech) solution at 68° C. for 90 minutes. Subsequently, the labeledDNA probe is added to the hybridization solution, and the probe is leftto hybridize to the membranes at 68° C. for 70 minutes. The membranesare washed five times in 2×SSC, 0.1% SDS at 42° C. for 5 minutes each,and finally washed for 30 minutes in 0.1×SSC, 0.2% SDS. Filters areexposed to Kodak XAR film (Eastman Kodak Company, Rochester, N.Y., USA)with an intensifying screen at −80° C. for 16 hours. One positive colonyis isolated from the plates, and replated with about 1000 pfu on a 15 cmLB plate. Plating, plaque lift to filters and hybridization areperformed as described above. About four positive phage plaques areisolated from this secondary screening.

[0264] cDNA containing plasmids (pBluescript SK-) are rescued from theisolated phages by in vivo excision by culturing XL-1 blue cellsco-infected with the isolated phages and with the Excision helper phage,as described by manufacturer (Stratagene). XL-blue cells containing theplasmids are plated on LB plates and grown at 37° C. for 16 hours.Colonies (18) from each plate are replated on LB plates and grown. Onecolony from each plate is stricken onto a nylon filter in an orderedarray, and the filter is placed on a LB plate to raise the colonies. Thefilter is then hybridized with a labeled probe as described above. Aboutthree positive colonies are selected and grown up in LB medium. PlasmidDNA is isolated from the three clones by Qiagen Midi Kit™ (Qiagen)according to the manufacturer's instructions. The size of the insert isdetermined by digesting the plasmid with the restriction enzymes NotIand SalI, which establishes an insert size. The sequence of the entireinsert is determined by automated sequencing on both strands of theplasmids.

EXAMPLE 3 Subcloning of the Coding Region of MMP via PCR

[0265] Additional experiments may be conducted to subclone the codingregion of MMP and place the isolated coding region into a useful vector.Two additional PCR primers are designed based on the coding region ofMMP, corresponding to either end. To protect against exonucleolyticattack during subsequent exposure to enzymes, e.g., Taq polymerase,primers are routinely synthesized with a protective run of nucleotidesat the 5′ end that are not necessarily complementary to the desiredtarget.

[0266] PCR is performed in a 50 μl reaction containing 34 μl H₂O, 5 μl10×buffer (140 mM ammonium sulfate, 0.1% gelatin, 0.6 M Tris-tricine, pH8.4), 5 μl 15 mM MgSO₄, 2 μl dNTP mixture (dGTP, dATP, dTTP, and dCTP,each at 10 mM), 3 μl genomic phage DNA (0.25 μg/μl), 0.3 μl Primer 1 (1μg/μl), 0.3 μl Primer 2 (1 μg/μl), 0.4 μl High Fidelity Taq polymerase(Boehringer Mannheim). The PCR reaction is started with 1 cycle of 94°C. for 2 minutes; followed by 25 cycles at 94° C. for 30 seconds, 55° C.for 30 seconds, and 72° C. for 1.3 minutes.

[0267] The contents from the PCR reaction are loaded onto a 2% agarosegel and fractionated. The DNA band of expected size is excised from thegel, placed in a GenElute Agarose spin column (Supelco), and spun for 10minutes at maximum speed in a microfuge tube placed in amicrocentrifuge. The eluted DNA is precipitated with ethanol andresuspended in 6 μl H₂O for ligation.

[0268] The PCR-amplified DNA fragment containing the coding region iscloned into pCR2.1 using a protocol standard in the art. In particular,the ligation reaction consists of 6 μl of MMP DNA, 1 μl 10×ligationbuffer, 2 μl pCR2.1 (25 ng/μl, Invitrogen), and 1 μl T4 DNA ligase(Invitrogen). The reaction mixture is incubated overnight at 14° C., andthe reaction is then terminated by heating at 65° C. for 10 minutes. Twomicroliters of the ligation reaction are transformed into One Shot cells(Invitrogen) and plated onto ampicillin plates. A single colonycontaining a recombinant pCR2.1 bearing an insert is used to inoculate a5 ml culture of LB medium. Plasmid DNA is purified using the ConcertRapid Plasmid Miniprep System (GibcoBRL) and sequenced. Followingconfirmation of the sequence, a 50 ml culture of LB medium is inoculatedwith the transformed One Shot cells, cultured, and processed using aQiagen Plasmid Midi Kit to yield purified pCR-MMP.

EXAMPLE 4 Hybridization Analysis to demonstrate MMP expression invarious tissues

[0269] The expression of MMP in mammals, such as the rat or mouse, isinvestigated by in situ hybridization histochemistry as described inBertilsson et al. (supra). Tissue sections are thaw-mounted ontosilanized, nuclease-free slides (CEL Associates, Inc., Houston, Tex.),and stored at −80° C. Sections are processed starting with post-fixationin cold 4% paraformaldehyde, rinsed in cold phosphate-buffered saline(PBS), acetylated using acetic anhydride in triethanolamine buffer, anddehydrated through a series of alcohol washes in 70%, 95%, and 100%alcohol at room temperature. Subsequently, sections are delipidated inchloroform, followed by rehydration through successive exposure to 100%and 95% alcohol at room temperature. Microscope slides containingprocessed cryosections are allowed to air dry prior to hybridization.

[0270] An MMP-specific probe is generated using PCR. Following PCRamplification, the fragment is digested with restriction enzymes andcloned into pBluescript II cleaved with the same enzymes. For productionof a probe specific for the sense strand of MMP, the MMP clone inpBluescript II is linearized with a suitable restriction enzyme, whichprovides a substrate for labeled run-off transcripts (i.e., cRNAriboprobes) using the vector-borne T7 promoter and commerciallyavailable T7 RNA polymerase. A probe specific for the antisense strandof MMP is also readily prepared using the MMP clone in pBluescript II bycleaving the recombinant plasmid with a suitable restriction enzyme togenerate a linearized substrate for the production of labeled run-offcRNA transcripts using the T3 promoter and cognate polymerase. Theriboprobes are labeled with [³⁵S]-UTP to yield a specific activity ofabout 0.40×10⁶ cpm/pmol for antisense riboprobes and about 0.65×10⁶cpm/pmol for sense-strand riboprobes Each riboprobe is subsequentlydenatured and added (2 pmol/ml) to hybridization buffer which contained50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA,1×Denhardt's Solution, and 10 mM dithiothreitol. Microscope slidescontaining sequential brain cryosections are independently exposed to 45μl of hybridization solution per slide and silanized cover slips areplaced over the sections being exposed to hybridization solution.Sections are incubated overnight (15-18 hours) at 52° C. to allowhybridization to occur. Equivalent series of cryosections are exposed tosense or antisense MMP-specific cRNA riboprobes.

[0271] Following the hybridization period, coverslips are washed off theslides in 1×SSC, followed by RNase A treatment involving the exposure ofslides to 20 μg/ml RNase A in a buffer containing 10 mM Tris-HCl (pH7.4), 0.5 M EDTA, and 0.5 M NaCl for 45 minutes at 37° C. Thecryosections are then subjected to three high-stringency washes in0.1×SSC at 52° C. for 20 minutes each. Following the series of washes,cryosections are dehydrated by consecutive exposure to 70%, 95%, and100% ammonium acetate in alcohol, followed by air drying and exposure toKodak BioMax MR-1 film. After 13 days of exposure, the film isdeveloped. Based on these results, slides containing tissue thathybridized, as shown by film autoradiograms, are coated with Kodak NTB-2nuclear track emulsion and the slides are stored in the dark for 32days. The slides are then developed and counterstained with hematoxylin.Emulsion-coated sections are analyzed microscopically to determine thespecificity of labeling. The signal is determined to be specific ifautoradiographic grains (generated by antisense probe hybridization) areclearly associated with cresyl violate-stained cell bodies.Autoradiographic grains found between cell bodies indicates non-specificbinding of the probe.

[0272] A PCR-based system (RapidScan™ Gene Expression Panel, OriGeneTechnologies, Rockville, Md.) may be used to generate a comprehensiveexpression profile of the putative MMP in human tissue, and in humanbrain regions. The RapidScan Expression Panel is comprised offirst-strand cDNAs from various human tissues and brain regions thatwere serially diluted over a 4-log range and arrayed into a multi-wellPCR plate. Human tissues arrayed may include: brain, heart, kidney,spleen, liver, colon, lung, small intestine, muscle, stomach, testis,placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary,uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.Human brain regions arrayed may include: frontal lobe, temporal lobe,cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala,thalamus, hypothalamus, pons, medulla and spinal cord.

[0273] Expression of the NMP in the various tissues is detected by usingPCR primers designed based on the available sequence of the protein thatwill prime the synthesis of a fragment of predetermined size in thepresence of the appropriate cDNA. The dilution range of cDNA depositedon the plate (e.g., 4-log) is chosen to ensure that the amplificationreaction is within the linear range and, hence, will facilitatesemi-quantitative determination of relative mRNA accumulation in thevarious tissues or brain regions examined.

[0274] Expression of MMP in different tissues provides an indicationthat modulators of MMP activity have utility for treating variousdisorders, including but not limited to metabolic diseases and disorders(e.g., type 2 diabetes, obesity, cardiovascular, dyslipidemias,adipogenesis, retinopathies, neuropathies, nephropathies etc.),proliferative diseases and cancers (e.g., different cancers such asbreast, colon, lung, etc., tumor growth, tumor invasion, andhyperproliferative disorders such as psoriasis, prostate hyperplasia,etc.), hormonal disorders (e.g., male/female hormonal replacement,polycystic ovarian syndrome, alopecia, etc.), CNS disorders (e.g.,degenerative disorders such as Parkinson's, Alzheimer's, etc.),inflammatory conditions (e.g., Chron's disease, arthritis), diseasesrelated to cell differentiation and homeostasis, cardiomyopathy,atherosclerosis, thromboembolic diseases, Sjögren's syndrome, renalfailure, periodontal diseases, retinal neovascularization, woundhealing, and neurodegenerative diseases including, for example,Alzheimer's disease, multiple sclerosis, Parkinson's disease, andmotoneuron disease, among others. Use of MMP modulators, including MMPligands (activators and repressors) and anti-MMP antibodies, to treatindividuals having such disease states is intended as an aspect of theinvention.

EXAMPLE 5 Northern Blot Analysis

[0275] Northern blots are performed to examine the expression of mRNA.The sense orientation oligonucleotide and the antisense-orientationoligonucleotide, described above, are used as primers to amplify aportion of the MMP cDNA sequence of a nucleotide sequence of SEQ ID NOS:1-3.

[0276] Multiple human tissue northern blot from Clontech are hybridizedwith the probe according to the recommendations of the manufacturer, andas described by Bertilsson (supra). The probe is labeled with α-³²P-dCTPby Rediprime™ DNA labeling system (Amersham Pharmacia), purified on NickColumn™ (Amersham Pharmacia) and added to the hybridization solution.The filters are washed several times at 42° C. in 0.2×SSC, 0.1% SDS.Filters are exposed to Kodak XAR film (Eastman Kodak Company, Rochester,N.Y., USA) with intensifying screen at −80° C.

EXAMPLE 6 Recombinant Expression of MMP in Eukaryotic Host Cells

[0277] A. Expression of MMP in Mammalian Cells

[0278] To produce MMP protein, a MMP-encoding polynucleotide isexpressed in a suitable host cell using a suitable expression vector andstandard genetic engineering techniques. For example, the MMP-encodingsequence described in Example 1 is subcloned into the commercialexpression vector pzeoSV2 (Invitrogen, San Diego, Cailf.) andtransfected into Chinese Hamster Ovary (CHO) cells using thetransfection reagent FuGENE 6 or Dosper (Boehringer-Mannheim) and thetransfection protocol provided in the product insert. Other eukaryoticcell lines, including human embryonic kidney (HEK 293), human coloncancer cells (CaCo-2), and COS cells, are suitable as well. Cells stablyexpressing MMP are selected by growth in the presence of 100 μg/mlzeocin (Stratagene, LaJolla, Calif.). Optionally, MMP may be purifiedfrom the cells using standard chromatographic techniques. To facilitatepurification, antisera is raised against one or more synthetic peptidesequences that correspond to portions of the MMP amino acid sequence,and the antisera is used to affinity purify MMP. The MMP also may beexpressed in-frame with a tag sequence (e.g., polyhistidine,hemagluttinin, FLAG) to facilitate purification. Moreover, it will beappreciated that many of the uses for MMP polypeptides, such as assaysdescribed below, do not require purification of MMP from the host cell.

[0279] B. Expression of MMP in HEK-293 cells

[0280] For expression of MMP in mammalian cells 293 (transformed human,primary embryonic kidney cells), a plasmid bearing the relevant MMPcoding sequence is prepared, using vector pSecTag2A (Invitrogen). VectorpSecTag2A contains the murine IgK chain leader sequence for secretion,the c-myc epitope for detection of the recombinant protein with theanti-myc antibody, a C-terminal polyhistidine for purification withnickel chelate chromatography, and a Zeocin resistant gene for selectionof stable transfectants. The forward primer for amplification of thisMMP cDNA is determined by routine procedures and preferably contains a5′ extension of nucleotides to introduce the HindIII cloning site andnucleotides matching the MMP sequence. The reverse primer is alsodetermined by routine procedures and preferably contains a 5′ extensionof nucleotides to introduce a XhoI restriction site for cloning andnucleotides corresponding to the reverse complement of the MMP sequence.The PCR conditions use 55° C. as the annealing temperature. The PCRproduct is gel purified and cloned into the HindIII-XhoI sites of thevector.

[0281] The DNA is purified using Qiagen chromatography columns andtransfected into 293 cells using DOTAP transfection media (BoehringerMannheim, Indianapolis, Ind.). Transiently transfected cells are testedfor expression after 24 hours of transfection, using western blotsprobed with anti-His and anti-MMP peptide antibodies. Permanentlytransfected cells are selected with Zeocin and propagated. Production ofthe recombinant protein is detected from both cells and media by westernblots probed with anti-His, anti-Myc or anti-MMP peptide antibodies.

[0282] C. Expression of MMP in COS cells

[0283] For expression of the MMP in COS7 cells, a polynucleotidemolecule having a nucleotide sequence of SEQ ID NOS: 1-3 can be clonedinto vector p3-CI. This vector is a pUC18-derived plasmid that containsthe HCMV (human cytomegalovirus) promoter-intron located upstream fromthe bGH (bovine growth hormone) polyadenylation sequence and a multiplecloning site. In addition, the plasmid contains the dhrf (dihydrofolatereductase) gene which provides selection in the presence of the drugmethotrexane (MTX) for selection of stable transformants.

[0284] The forward primer is determined by routine procedures andpreferably contains a 5′ extension which introduces an XbaI restrictionsite for cloning, followed by nucleotides which correspond to anucleotide sequence of SEQ ID NOS: 1-3. The reverse primer is alsodetermined by routine procedures and preferably contains 5′- extensionof nucleotides which introduces a SalI cloning site followed bynucleotides which correspond to the reverse complement of a nucleotidesequence of SEQ ID NOS: 1-3.

[0285] The PCR consists of an initial denaturation step of 5 min at 95°C., 30 cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58°C. and 30 sec extension at 72° C., followed by 5 min extension at 72° C.The PCR product is gel purified and ligated into the XbaI and SalI sitesof vector p3-CI. This construct is transformed into E. coli cells foramplification and DNA purification. The DNA is purified with Qiagenchromatography columns and transfected into COS 7 cells usingLipofectamine reagent from BRL, following the manufacturer's protocols.Forty-eight and 72 hours after transfection, the media and the cells aretested for recombinant protein expression.

[0286] MMP expressed from a COS cell culture can be purified byconcentrating the cell-growth media to about 10 mg of protein/ml, andpurifying the protein by, for example, chromatography. Purified MMP isconcentrated to 0.5 mg/ml in an Amicon concentrator fitted with a YM-10membrane and stored at −80° C.

[0287] D. Expression of MMP in Insect Cells

[0288] For expression of MMP in a baculovirus system, a polynucleotidemolecule having a nucleotide sequence of SEQ ID NOS: 1-3 can beamplified by PCR. The forward primer is determined by routine proceduresand preferably contains a 5′ extension which adds the NdeI cloning site,followed by followed by nucleotides which correspond to a nucleotidesequence of SEQ ID NOS: 1-3. The reverse primer is also determined byroutine procedures and preferably contains a 5′ extension whichintroduces the KpnI cloning site, followed by followed by nucleotideswhich correspond to the reverse complement of a nucleotide sequence ofSEQ ID NOS: 1-3.

[0289] The PCR product is gel purified, digested with NdeI and KpnI, andcloned into the corresponding sites of vector pACHTL-A (Pharmingen, SanDiego, Calif.). The pAcHTL expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV), and a 6×His tag upstream from the multiple cloning site.A protein kinase site for phosphorylation and a thrombin site forexcision of the recombinant protein precede the multiple cloning site isalso present. Of course, many other baculovirus vectors could be used inplace of pAcHTL-A, such as pAc373, pVL941 and pAcIM1. Other suitablevectors for the expression of MMP polypeptides can be used, providedthat the vector construct includes appropriately located signals fortranscription, translation, and trafficking, such as an in-frame AUG anda signal peptide, as required. Such vectors are described in Luckow etal., Virology 170:31-39, among others.

[0290] The virus is grown and isolated using standard baculovirusexpression methods, such as those described in Summers et al. (A Manualof Methods for Baculovirus Vectors and Insect Cell Culture Procedures,Texas Agricultural Experimental Station Bulletin No. 1555 (1987)).

[0291] In a preferred embodiment, pAcHLT-A containing MMP gene isintroduced into baculovirus using the “BaculoGold” transfection kit(Pharmingen, San Diego, Calif.) using methods established by themanufacturer. Individual virus isolates are analyzed for proteinproduction by radiolabeling infected cells with ³⁵S-methionine at 24hours post infection. Infected cells are harvested at 48 hours postinfection, and the labeled proteins are visualized by SDS-PAGE. Virusesexhibiting high expression levels can be isolated and used for scaled upexpression.

[0292] For expression of a MMP polypeptide in Sf9 cells, apolynucleotide molecule having a nucleotide sequence of SEQ ID NOS: 1-3,can be amplified by PCR using the primers and methods described abovefor baculovirus expression. The MMP cDNA is cloned into vector pAcHLT-A(Pharmingen) for expression in Sf9 insect. The insert is cloned into theNdeI and KpnI sites, after elimination of an internal NdeI site (usingthe same primers described above for expression in baculovirus). DNA ispurified with Qiagen chromatography columns and expressed in Sf9 cells.Preliminary Western blot experiments from non purified plaques aretested for the presence of the recombinant protein of the expected sizewhich reacted with the MMP-specific antibody. These results areconfirmed after further purification and expression optimization in HiG5cells.

EXAMPLE 7 Zymography

[0293] MMP protease activity is analyzed by substrate gelelectrophoresis (zymography) in polyacrylamide gels containing 2 mg/mlgelatin or 1.5 mg/ml casein. Samples are dissolved in modified Laemmlisample buffer [containing 2.5% (v/v) SDS without 2-mercaptoethanol] andelectrophoresed, without prior boiling, at 4° C. After removal of theSDS by washing in 2.5% (v/v) Triton X-100 in 50 mM Tris/HCl, pH 7.5, for1 h, the gels are incubated overnight at 37° C. in a buffer containing40 mM Tris/HCl, pH 7.5, and 10 mM CaCl₂. Staining with 0.5% (w/v)Coomassie Brilliant Blue (Bio-Rad, Richmond, Calif., U.S.A.) in 30%(v/v) isopropanol/10% (v/v) acetic acid followed by destaining with 30%isopropanol/10% acetic acid allows identification of gelatinolytic orcaseinolytic activity as clear zones in a blue background.

EXAMPLE 8 Antibodies to MMP

[0294] Standard techniques are employed to generate polyclonal ormonoclonal antibodies to the MMP, and to generate useful antigen-bindingfragments thereof or variants thereof, including “humanized” variants.Such protocols can be found, for example, in Sambrook et al. (1989) andHarlow et al. (Eds.), Antibodies A Laboratory Manual; Cold Spring HarborLaboratory; Cold Spring Harbor, N.Y. (1988). In one embodiment,recombinant MMP polypeptides (or cells or cell membranes containing suchpolypeptides) are used as antigen to generate the antibodies. In anotherembodiment, one or more peptides having amino acid sequencescorresponding to an immunogenic portion of MMP (e.g., 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) are used asantigen. The antigen may be mixed with an adjuvant or linked to a haptento increase antibody production.

[0295] A. Polyclonal or Monoclonal antibodies

[0296] As one exemplary protocol, recombinant MMP or a syntheticfragment thereof is used to immunize a mouse for generation ofmonoclonal antibodies (or larger mammal, such as a rabbit, forpolyclonal antibodies). To increase antigenicity, peptides areconjugated to Keyhole Lympet Hemocyanin (Pierce), according to themanufacturer's recommendations. For an initial injection, the antigen isemulsified with Freund's Complete Adjuvant and injected subcutaneously.At intervals of two to three weeks, additional aliquots of MMP antigenare emulsified with Freund's Incomplete Adjuvant and injectedsubcutaneously. Prior to the final booster injection, a serum sample istaken from the immunized mice and assayed by western blot to confirm thepresence of antibodies that immunoreact with MMP. Serum from theimmunized animals may be used as a polyclonal antisera or used toisolate polyclonal antibodies that recognize MMP. Alternatively, themice are sacrificed and their spleen removed for generation ofmonoclonal antibodies.

[0297] To generate monoclonal antibodies, the spleens are placed in 10ml serum-free RPMI 1640, and single cell suspensions are formed bygrinding the spleens in serum-free RPMI 1640, supplemented with 2 mML-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100μg/ml streptomycin (RPMI) (Gibco, Canada). The cell suspensions arefiltered and washed by centrifugation and resuspended in serum-freeRPMI. Thymocytes taken from three naive Balb/c mice are prepared in asimilar manner and used as a Feeder Layer. NS-1 myeloma cells, kept inlog phase in RPMI with 10% fetal bovine serum (FBS) (HycloneLaboratories, Inc., Logan, Utah) for three days prior to fusion, arecentrifuged and washed as well.

[0298] To produce hybridoma fusions, spleen cells from the immunizedmice are combined with NS-1 cells and centrifuged, and the supernatantis aspirated. The cell pellet is dislodged by tapping the tube, and 2 mlof 37° C. PEG 1500 (50% in 75 mM BEPES, pH 8.0) (Boehringer-Mannheim) isstirred into the pellet, followed by the addition of serum-free RPMI.Thereafter, the cells are centrifuged, resuspended in RPMI containing15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine(HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×10⁶thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissueculture plates (Corning, Corning N.Y.).

[0299] On days 2, 4, and 6 after the fusion, 100 μl of medium is removedfrom the wells of the fusion plates and replaced with fresh medium. Onday 8, the fusions are screened by ELISA, testing for the presence ofmouse IgG that binds to MMP. Selected fusion wells are further cloned bydilution until monoclonal cultures producing anti-MMP antibodies areobtained.

[0300] B. Humanization of anti-MMP Monoclonal Antibodies

[0301] The expression pattern of MMP as reported herein suggeststherapeutic indications for MMP inhibitors (repressors).MMP-neutralizing antibodies comprise one class of therapeutics useful asMMP repressors. Following are protocols to improve the utility ofanti-MMP monoclonal antibodies as therapeutics in humans by “humanizing”the monoclonal antibodies to improve their serum half-life and renderthem less immunogenic in human hosts (i.e., to prevent human antibodyresponse to non-human anti-MMP antibodies).

[0302] The principles of humanization have been described in theliterature and are facilitated by the modular arrangement of antibodyproteins. To minimize the possibility of binding complement, a humanizedantibody of the IgG4 isotype is preferred.

[0303] For example, a level of humanization is achieved by generatingchimeric antibodies comprising the variable domains of non-humanantibody proteins of interest with the constant domains of humanantibody molecules. (See, e.g., Morrison et al., Adv. Immunol., 44:65-92(1989)). The variable domains of MMP-neutralizing anti-MMP antibodiesare cloned from the genomic DNA of a B-cell hybridoma or from cDNAgenerated from mRNA isolated from the hybridoma of interest. The Vregion gene fragments are linked to exons encoding human antibodyconstant domains, and the resultant construct is expressed in suitablemammalian host cells (e.g., myeloma or CHO cells).

[0304] To achieve an even greater level of humanization, only thoseportions of the variable region gene fragments that encodeantigen-binding complementarity determining regions (CDR) of thenon-human monoclonal antibody genes are cloned into human antibodysequences. (See, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-36 (1988); and Tempest et al., Bio/Technology 9:266-71 (1991)).If necessary, the β-sheet framework of the human antibody surroundingthe CDR3 regions also is modified to more closely mirror the threedimensional structure of the antigen-binding domain of the originalmonoclonal antibody. (See Kettleborough et al., Protein Engin.,4:773-783 (1991); and Foote et al., J. Mol. Biol., 224:487-499 (1992)).

[0305] In an alternative approach, the surface of a non-human monoclonalantibody of interest is humanized by altering selected surface residuesof the non-human antibody, e.g., by site-directed mutagenesis, whileretaining all of the interior and contacting residues of the non-humanantibody. See Padlan, Molecular Immunol., 28(4/5):489-98 (1991).

[0306] The foregoing approaches are employed using MMP-neutralizinganti-MMP monoclonal antibodies and the hybridomas that produce them togenerate humanized MMP-neutralizing antibodies that are useful astherapeutics to treat or palliate conditions wherein MMP expression orligand-mediated MMP activity is detrimental.

[0307] C. Human MMP-Neutralizing Antibodies from Phage Display

[0308] Human MMP-neutralizing antibodies are generated by phage displaytechniques such as those described in Aujame et al., Human Antibodies8(4): 155-168 (1997); Hoogenboom, TIBTECH 15:62-70 (1997); and Rader etal., Curr. Opin. Biotechnol. 8:503-508 (1997), all of which areincorporated by reference. For example, antibody variable regions in theform of Fab fragments or linked single chain Fv fragments are fused tothe amino terminus of filamentous phage minor coat protein pIII.Expression of the fusion protein and incorporation thereof into themature phage coat results in phage particles that present an antibody ontheir surface and contain the genetic material encoding the antibody. Aphage library comprising such constructs is expressed in bacteria, andthe library is screened for MMP-specific phage-antibodies using labeledor immobilized MMP as antigen-probe.

[0309] D. Human MMP-neutralizing Antibodies from Transgenic Mice

[0310] Human NMP-neutralizing antibodies are generated in transgenicmice essentially as described in Bruggemann et al., Immunol. Today17(8):391-97 (1996) and Bruggemann et al., Curr. Opin. Biotechnol.8:455-58 (1997). Transgenic mice carrying human V-gene segments ingermline configuration and that express these transgenes in theirlymphoid tissue are immunized with a MMP composition using conventionalimmunization protocols. Hybridomas are generated using B cells from theimmunized mice using conventional protocols and screened to identifyhybridomas secreting anti-MMP human antibodies (e.g., as describedabove).

EXAMPLE 9 Assays to Assess MMP Activity and to Identify Modulators ofMMP Activity

[0311] A. Synthetic Fluorogenic Peptide Substrate Cleavage Assays

[0312] Fluorogenic peptide substrates may be used to test proteinaseactivity as well as inhibitors of proteinase activity. Such peptidesubstrates may be purchased, for example, from BACHEM Bioscience Inc,who offer several aMP substrates and various enzyme substrates, forexample a substrate of tumor necrosis factor-α (TNF-α) converting enzyme(TACE). Substrates are prepared as 50-500 μM stock solutions in 1:1dimethyl sulfoxide (DMSO) and water. Fluorescent assays are performed atλ_(excitation)=328 nm and λ_(emmission)=393 nm using a luminescencespectrometer equipped with a constant-temperature water bath. Therelationship between fluorescence units and nanomoles of productproduced is determined from the fluorescence value obtained when all thesubstrate is hydrolyzed.

[0313] B. Kinetic Parameters

[0314] Assays for obtaining kinetic parameters are performed at 25° C.in 10 mM CaCl₂ 0.2 M NaCl and 0.05% Brij-35 in 50 mM BEPES, pH 7.5 overthe substrate concentration range 1-4 μM range and enzyme concentrationrange 0.06-50 nM under steady-state conditions. Stock solutions of MMPsare diluted to 1-500 nM by adding 50 mM HEPES buffer containing 10 mMCaCl₂ 0.2 M NaCl, and 0.05% Brij-35 or 50 mM Tricine buffer with thesame constituents. A typical assay is carried out by incubating 186 μLof buffer solution and 4 μL of substrate solution in an assay cuvettefor at least for 15 min. at 25° C., and then adding 10 μL of enzymesolution into the assay cuvette. Initial hydrolysis rates are monitoredfor 10-30 min.

[0315] C. Inhibition Studies

[0316] Modulators of MMP activity may be studied using the above assay.The activity of MMPs in the absence of potential modulating compounds iscompared to the activity of MMPs in the presence of varyingconcentrations of potential modulating compounds.

[0317] Among the modulators that can be identified by these assays arenatural ligand compounds of the MMP; synthetic analogs and derivativesof natural ligands; antibodies, antibody fragments, and/or antibody-likecompounds derived from natural antibodies or from antibody-likecombinatorial libraries; and/or synthetic compounds identified byhigh-throughput screening of libraries; and the like. All modulatorsthat bind MMP are useful for identifying MMPs in tissue samples (e.g.,for diagnostic purposes, pathological purposes, and the like). Activatorand repressor modulators are useful for up-regulating anddown-regulating MMP activity, respectively, to treat disease statescharacterized by abnormal levels of MMP activity. The assays may beperformed using single putative modulators, and/or may be performedusing a known activator in combination with candidate repressors (orvisa versa).

[0318] Some of the preferred embodiments of the invention describedabove are outlined below and include, but are not limited to, thefollowing embodiments. As those skilled in the art will appreciate,numerous changes and modifications may be made to the preferredembodiments of the invention without departing from the spirit of theinvention. It is intended that all such variations fall within the scopeof the invention.

[0319] The entire disclosure of each publication cited herein is herebyincorporated by reference.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 8 <210> SEQ ID NO 1 <211>LENGTH: 1845 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 1 gcttcagctg aagaaagaga ggaatgaagc gccttctgct tctgtgtttgttctttataa 60 cattttcttc tgcatttccc ttagtccgga tgacggaaaa tgaagaaaatatgcaactgg 120 ctcaggcata tctcaaccag ttctactctc ttgaaataga agggaatcatcttgttcaaa 180 gcaagaatag gagtctcata gatgacaaaa ttcgggaaat gcaagcattttttggattga 240 cagtgactgg aaaactggac tcaaacaccc ttgagatcat gaagacacccaggtgtgggg 300 tgcctgatgt gggccagtat ggctacaccc tccctgggtg gagaaaatacaacctcacct 360 acagaataat aaactatact ccggatatgg cacgagctgc tgtggatgaggctatccaag 420 aaggtttaga agtgtggagc aaagtcactc cactaaaatt caccaagatttcaaagggga 480 ttgcagacat catgattgcc tttaggactc gagtccatgg tcggtgtcctcgctattttg 540 atggtccctt gggagtgctt ggccatgcct ttcctcctgg tccgggtctgggtggtgaca 600 ctcattttga tgaggatgaa aactggacca aggatggagc aggattcaacttgtttcttg 660 tggctgctca tgaatttggt catgcactgg ggctctctca ctccaatgatcaaacagcct 720 tgatgttccc aaattatgtc tccctggatc ccagaaaata cccactttctcaggatgata 780 tcaatggaat ccagtccatc tatggaggtc tgcctaaggt acctgctaagccaaaggaac 840 ccactatacc ccatgcctgt gaccctgact tgacttttga cgctatcacaactttccgca 900 gagaagtaat gttctttaaa ggcaggcacc tatggaggat ctattatgatatcacggatg 960 ttgagtttga attaattgct tcattctggc catctctgcc agctgatctgcaagctgcat 1020 acgagaaccc cagagataag attctggttt ttaaagatga aaacttctggatgatcagag 1080 gatatgctgt cttgccagat tatcccaaat ccatccatac attaggttttccaggacgtg 1140 tgaagaaaat agatgcagcc gtctgtgata agaccacaag aaaaacctacttctttgtgg 1200 gcatttggtg ctggaggttt gatgaaatga cccaaaccat ggacaaaggattcccgcaga 1260 gagtggtaaa acactttcct ggaatcagta tccgtgttga tgctgctttccagtacaaag 1320 gattcttctt tttcagccgt ggatcaaagc aatttgaata caacattaagacaaagaata 1380 ttacccgaat catgagaact aatacttggt ttcaatgcaa agaaccaaagaactcctcat 1440 ttggttttga tatcaacaag gaaaaagcac attcaggagg cataaagatattgtatcata 1500 agagtttaag cttgtttatt tttggtattg ttcatttgct gaaaaacacttctatttatc 1560 aataaattca tagacctaaa ataaacctca acaggtcttt taatataaattctgcttcaa 1620 aatagaataa aaccattctt taacaacaag ttgctggtcc tagttctaaatatccaaatt 1680 caatggccat tttgagctgc ctgattcttt taataggaag ttattatgtagaaacaaaaa 1740 tctctgactg tactttaagc ctatttcatg ctttgtggac ttggagaagacatgtcttat 1800 aactgaatac tgaaacattt attaaaccaa tctttagcat tctaa 1845<210> SEQ ID NO 2 <211> LENGTH: 989 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 2 gacaaatgag ggtttggcat gcagctcgtc atcttaagagttactatctt cttgccctgg 60 tgtttcgccg ttccagtgcc ccctgctgca gaccataaaggatgggactt tgttgagggc 120 tatttccatc aatttttcct gaccaagaag gagtcgccactccttaccca ggagacacaa 180 acacagctcc tgcaacaatt ccatcggaat gggacagacctacttgacat gcagatgcat 240 gctctgctac accagcccca ctgtggggtg cctgatgggtccgacacctc catctcgcca 300 ggaagatgca agtggaataa gcacactcta acttacaggattatcaatta cccacatgat 360 atgaagccat ccgcagtgaa agacagtata tataatgcagtttccatctg gagcaatgtg 420 acccctttga tattccagca agtgcagaat ggagatgcagacatcaaggt ttctttctgg 480 cagtgggccc atgaagatgg ttggcccttt gatgggccaggtggtatctt aggccatgcc 540 tttttaccaa attctggaaa tcctggagtt gtccattttgacaagaatga acactggtca 600 gcttcagaca ctggatataa tctgttcctg gttgcaactcatgagattgg gcattctttg 660 ggcctgcagc actctgggaa tcagagctcc ataatgtaccccacttactg gtatcacgac 720 cctagaacct tccagctcag tgccgatgat atccaaaggatccagcattt gtatggagaa 780 aaatgttcat ctgacatacc ttaatgttag cacagaggacttattcaacc tgtcctttca 840 gggagtttat tggaggatca aagaactgaa agcactagagcagccttggg gactgctagg 900 atgaagccct aaagaatgca acctagtcag gttagctgaaccgacactca aaacgctact 960 gagtcacaat aaagattgtt ttaaagagt 989 <210> SEQID NO 3 <211> LENGTH: 1597 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 3 gctccccgag ccgggctgca ccggaggcgg cgagatggtc gcgcgcgtcggcctcctgct 60 gcgcgccctg cagctgctac tgtggggcca cctggacgcc cagcccgcggagcgcggagg 120 ccaggagctg cgcaaggagg cggaggcatt cctagagaag tacggatacctcaatgaaca 180 ggtccccaaa gctcccacct ccactcgatt cagcgatgcc atcagagcgtttcagtgggt 240 gtcccagcta cctgtcagcg gcgtgttgga ccgcgccacc ctgcgccagatgactcgtcc 300 ccgctgcggg gttacagata ccaacagtta tgcggcctgg gctgagaggatcagtgactt 360 gtttgctaga caccggacca aaatgaggcg taagaaacgc tttgcaaagcaaggtaacaa 420 atggtacaag cagcacctct cctaccgcct ggtgaactgg cctgagcatctccggagccg 480 gcagttcggg gcgccgtgcg cgccgccttc cagttgtgga gcaacgtctcagcgctggag 540 ttctgggagg ccccagccac aggccccgct gacatccggc tcaccttcttccaaggggac 600 cacaacgatg ggctgggcaa tgcctttgat ggcccagggg gcgccctggcgcacgccttt 660 cctgccccgc cgcggcgaag cgcacttcga ccaagatgag cgctggtccctgagccgccg 720 ccgcgggcgc aacctgttcg tggtgctggc gcacgagatc ggtcacacgcttggcctcac 780 ccactcgccc gcgccgcgcg cgctcatggc gccctactac aagaggctgggccgcgacgc 840 gctgctcagc tgggacgacg tgctggccgt gcagagcctg tatgggaagcccctaggggg 900 ctcagtggcc gtccagctcc caggaaagct gttcactgac tttgagacctgggactccta 960 cagcccccaa ggaaggcgcc ctgaaacgca gggccctaaa tactgccactcttccttcga 1020 tgccatcact gtagacaggc aacagcaact gtacattttt aaagggagccatttctggga 1080 ggtggcagct gatggcaacg tctcagagcc ccgtccactg caggaaagatgggtcgggct 1140 gccccccaac attgaggctg cggcagtgtc attgaatgat ggagatttctacttcttcaa 1200 agggggtcga tgctggaggt tccggggccc caagccagtg tggggtctcccacagctgtg 1260 ccgggcaggg ggcctgcccc gccatcctga cgccgccctc ttcttccctcctctgcgccg 1320 cctcatcctc ttcaagggtg cccgctacta cgtgctggcc cgagggggactgcaagtgga 1380 gccctactac ccccgaagtc tgcaggactg gggaggcatc cctgaggaggtcagcggcgc 1440 cctgccgagg cccgatggct ccatcatctt cttccgagat gaccgctactggcgcctcga 1500 ccaggccaaa ctgcaggcaa ccacctcggg ccgctgggcc accgagctgccctggatggg 1560 ctgctggcat gccaactcgg ggagcgccct gttctga 1597 <210> SEQID NO 4 <211> LENGTH: 513 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 4 Met Lys Arg Leu Leu Leu Leu Cys Leu Phe Phe Ile ThrPhe Ser Ser 1 5 10 15 Ala Phe Pro Leu Val Arg Met Thr Glu Asn Glu GluAsn Met Gln Leu 20 25 30 Ala Gln Ala Tyr Leu Asn Gln Phe Tyr Ser Leu GluIle Glu Gly Asn 35 40 45 His Leu Val Gln Ser Lys Asn Arg Ser Leu Ile AspAsp Lys Ile Arg 50 55 60 Glu Met Gln Ala Phe Phe Gly Leu Thr Val Thr GlyLys Leu Asp Ser 65 70 75 80 Asn Thr Leu Glu Ile Met Lys Thr Pro Arg CysGly Val Pro Asp Val 85 90 95 Gly Gln Tyr Gly Tyr Thr Leu Pro Gly Trp ArgLys Tyr Asn Leu Thr 100 105 110 Tyr Arg Ile Ile Asn Tyr Thr Pro Asp MetAla Arg Ala Ala Val Asp 115 120 125 Glu Ala Ile Gln Glu Gly Leu Glu ValTrp Ser Lys Val Thr Pro Leu 130 135 140 Lys Phe Thr Lys Ile Ser Lys GlyIle Ala Asp Ile Met Ile Ala Phe 145 150 155 160 Arg Thr Arg Val His GlyArg Cys Pro Arg Tyr Phe Asp Gly Pro Leu 165 170 175 Gly Val Leu Gly HisAla Phe Pro Pro Gly Pro Gly Leu Gly Gly Asp 180 185 190 Thr His Phe AspGlu Asp Glu Asn Trp Thr Lys Asp Gly Ala Gly Phe 195 200 205 Asn Leu PheLeu Val Ala Ala His Glu Phe Gly His Ala Leu Gly Leu 210 215 220 Ser HisSer Asn Asp Gln Thr Ala Leu Met Phe Pro Asn Tyr Val Ser 225 230 235 240Leu Asp Pro Arg Lys Tyr Pro Leu Ser Gln Asp Asp Ile Asn Gly Ile 245 250255 Gln Ser Ile Tyr Gly Gly Leu Pro Lys Val Pro Ala Lys Pro Lys Glu 260265 270 Pro Thr Ile Pro His Ala Cys Asp Pro Asp Leu Thr Phe Asp Ala Ile275 280 285 Thr Thr Phe Arg Arg Glu Val Met Phe Phe Lys Gly Arg His LeuTrp 290 295 300 Arg Ile Tyr Tyr Asp Ile Thr Asp Val Glu Phe Glu Leu IleAla Ser 305 310 315 320 Phe Trp Pro Ser Leu Pro Ala Asp Leu Gln Ala AlaTyr Glu Asn Pro 325 330 335 Arg Asp Lys Ile Leu Val Phe Lys Asp Glu AsnPhe Trp Met Ile Arg 340 345 350 Gly Tyr Ala Val Leu Pro Asp Tyr Pro LysSer Ile His Thr Leu Gly 355 360 365 Phe Pro Gly Arg Val Lys Lys Ile AspAla Ala Val Cys Asp Lys Thr 370 375 380 Thr Arg Lys Thr Tyr Phe Phe ValGly Ile Trp Cys Trp Arg Phe Asp 385 390 395 400 Glu Met Thr Gln Thr MetAsp Lys Gly Phe Pro Gln Arg Val Val Lys 405 410 415 His Phe Pro Gly IleSer Ile Arg Val Asp Ala Ala Phe Gln Tyr Lys 420 425 430 Gly Phe Phe PhePhe Ser Arg Gly Ser Lys Gln Phe Glu Tyr Asn Ile 435 440 445 Lys Thr LysAsn Ile Thr Arg Ile Met Arg Thr Asn Thr Trp Phe Gln 450 455 460 Cys LysGlu Pro Lys Asn Ser Ser Phe Gly Phe Asp Ile Asn Lys Glu 465 470 475 480Lys Ala His Ser Gly Gly Ile Lys Ile Leu Tyr His Lys Ser Leu Ser 485 490495 Leu Phe Ile Phe Gly Ile Val His Leu Leu Lys Asn Thr Ser Ile Tyr 500505 510 Gln <210> SEQ ID NO 5 <211> LENGTH: 259 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 5 Met Gln Leu Val Ile Leu Arg ValThr Ile Phe Leu Pro Trp Cys Phe 1 5 10 15 Ala Val Pro Val Pro Pro AlaAla Asp His Lys Gly Trp Asp Phe Val 20 25 30 Glu Gly Tyr Phe His Gln PhePhe Leu Thr Lys Lys Glu Ser Pro Leu 35 40 45 Leu Thr Gln Glu Thr Gln ThrGln Leu Leu Gln Gln Phe His Arg Asn 50 55 60 Gly Thr Asp Leu Leu Asp MetGln Met His Ala Leu Leu His Gln Pro 65 70 75 80 His Cys Gly Val Pro AspGly Ser Asp Thr Ser Ile Ser Pro Gly Arg 85 90 95 Cys Lys Trp Asn Lys HisThr Leu Thr Tyr Arg Ile Ile Asn Tyr Pro 100 105 110 His Asp Met Lys ProSer Ala Val Lys Asp Ser Ile Tyr Asn Ala Val 115 120 125 Ser Ile Trp SerAsn Val Thr Pro Leu Ile Phe Gln Gln Val Gln Asn 130 135 140 Gly Asp AlaAsp Ile Lys Val Ser Phe Trp Gln Trp Ala His Glu Asp 145 150 155 160 GlyTrp Pro Phe Asp Gly Pro Gly Gly Ile Leu Gly His Ala Phe Leu 165 170 175Pro Asn Ser Gly Asn Pro Gly Val Val His Phe Asp Lys Asn Glu His 180 185190 Trp Ser Ala Ser Asp Thr Gly Tyr Asn Leu Phe Leu Val Ala Thr His 195200 205 Glu Ile Gly His Ser Leu Gly Leu Gln His Ser Gly Asn Gln Ser Ser210 215 220 Ile Met Tyr Pro Thr Tyr Trp Tyr His Asp Pro Arg Thr Phe GlnLeu 225 230 235 240 Ser Ala Asp Asp Ile Gln Arg Ile Gln His Leu Tyr GlyGlu Lys Cys 245 250 255 Ser Ser Asp <210> SEQ ID NO 6 <211> LENGTH: 520<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 6 Met ValAla Arg Val Gly Leu Leu Leu Arg Ala Leu Gln Leu Leu Leu 1 5 10 15 TrpGly His Leu Asp Ala Gln Pro Ala Glu Arg Gly Gly Gln Glu Leu 20 25 30 ArgLys Glu Ala Glu Ala Phe Leu Glu Lys Tyr Gly Tyr Leu Asn Glu 35 40 45 GlnVal Pro Lys Ala Pro Thr Ser Thr Arg Phe Ser Asp Ala Ile Arg 50 55 60 AlaPhe Gln Trp Val Ser Gln Leu Pro Val Ser Gly Val Leu Asp Arg 65 70 75 80Ala Thr Leu Arg Gln Met Thr Arg Pro Arg Cys Gly Val Thr Asp Thr 85 90 95Asn Ser Tyr Ala Ala Trp Ala Glu Arg Ile Ser Asp Leu Phe Ala Arg 100 105110 His Arg Thr Lys Met Arg Arg Lys Lys Arg Phe Ala Lys Gln Gly Asn 115120 125 Lys Trp Tyr Lys Gln His Leu Ser Tyr Arg Leu Val Asn Trp Pro Glu130 135 140 His Leu Arg Ser Arg Gln Phe Gly Ala Pro Cys Ala Pro Pro SerSer 145 150 155 160 Cys Gly Ala Thr Ser Gln Arg Trp Ser Ser Gly Arg ProGln Pro Gln 165 170 175 Ala Pro Leu Thr Ser Gly Ser Pro Ser Ser Lys GlyThr Thr Thr Met 180 185 190 Gly Trp Ala Met Pro Leu Met Ala Gln Gly AlaPro Trp Arg Thr Pro 195 200 205 Phe Leu Pro Arg Arg Gly Glu Ala His PheAsp Gln Asp Glu Arg Trp 210 215 220 Ser Leu Ser Arg Arg Arg Gly Arg AsnLeu Phe Val Val Leu Ala His 225 230 235 240 Glu Ile Gly His Thr Leu GlyLeu Thr His Ser Pro Ala Pro Arg Ala 245 250 255 Leu Met Ala Pro Tyr TyrLys Arg Leu Gly Arg Asp Ala Leu Leu Ser 260 265 270 Trp Asp Asp Val LeuAla Val Gln Ser Leu Tyr Gly Lys Pro Leu Gly 275 280 285 Gly Ser Val AlaVal Gln Leu Pro Gly Lys Leu Phe Thr Asp Phe Glu 290 295 300 Thr Trp AspSer Tyr Ser Pro Gln Gly Arg Arg Pro Glu Thr Gln Gly 305 310 315 320 ProLys Tyr Cys His Ser Ser Phe Asp Ala Ile Thr Val Asp Arg Gln 325 330 335Gln Gln Leu Tyr Ile Phe Lys Gly Ser His Phe Trp Glu Val Ala Ala 340 345350 Asp Gly Asn Val Ser Glu Pro Arg Pro Leu Gln Glu Arg Trp Val Gly 355360 365 Leu Pro Pro Asn Ile Glu Ala Ala Ala Val Ser Leu Asn Asp Gly Asp370 375 380 Phe Tyr Phe Phe Lys Gly Gly Arg Cys Trp Arg Phe Arg Gly ProLys 385 390 395 400 Pro Val Trp Gly Leu Pro Gln Leu Cys Arg Ala Gly GlyLeu Pro Arg 405 410 415 His Pro Asp Ala Ala Leu Phe Phe Pro Pro Leu ArgArg Leu Ile Leu 420 425 430 Phe Lys Gly Ala Arg Tyr Tyr Val Leu Ala ArgGly Gly Leu Gln Val 435 440 445 Glu Pro Tyr Tyr Pro Arg Ser Leu Gln AspTrp Gly Gly Ile Pro Glu 450 455 460 Glu Val Ser Gly Ala Leu Pro Arg ProAsp Gly Ser Ile Ile Phe Phe 465 470 475 480 Arg Asp Asp Arg Tyr Trp ArgLeu Asp Gln Ala Lys Leu Gln Ala Thr 485 490 495 Thr Ser Gly Arg Trp AlaThr Glu Leu Pro Trp Met Gly Cys Trp His 500 505 510 Ala Asn Ser Gly SerAla Leu Phe 515 520 <210> SEQ ID NO 7 <211> LENGTH: 999 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <301>AUTHORS: Park, H.I., Ni, J., Gerkema, F.E., Liu, D., Belozero Sang, Q.X.<302> TITLE: Identification and characterization of humanendometalloproteinase-26) from endometrial tumor <303> JOURNAL: J. Biol.Chem. <304> VOLUME: 275 <305> ISSUE: 27 <306> PAGES: 20540-20544 <307>DATE: 2000-03-23 <308> DATABASE ACCESSION NUMBER: GenBankAF248646 <309>DATABASE ENTRY DATE: 2000-03-23 <300> PUBLICATION INFORMATION: <308>DATABASE ACCESSION NUMBER: GenbankAF248646 <309> DATABASE ENTRY DATE:2000-03-23 <400> SEQUENCE: 7 ggcacgagca tgcagctcgt catcttaaga gttactatcttcttgccctg gtgtttcgcc 60 gttccagtgc cccctgctgc agaccataaa ggatgggactttgttgaggg ctatttccat 120 caatttttcc tgaccgagaa ggagtcgcca ctccttacccaggagacaca aacacagctc 180 ctgcaacaat tccatcggaa tgggacagac ctacttgacatgcagatgca tgctctgcta 240 caccagcccc actgtggggt gcctgatggg tccgacacctccatctcgcc aggaagatgc 300 aagtggaata agcacactct aacttacagg attatcaattacccacatga tatgaagcca 360 tccgcagtga aagacagtat atataatgca gtttccatctggagcaatgt gacccctttg 420 atattccagc aagtgcagaa tggagatgca gacatcaaggtttctttctg gcagtgggcc 480 catgaagatg gttggccctt tgatgggcca ggtggtatcttaggccatgc ctttttacca 540 aattctggaa atcctggagt tgtccatttt gacaagaatgaacactggtc agcttcagac 600 actggatata atctgttcct ggttgcaact catgagattgggcattcttt gggcctgcag 660 cactctggga atcagagctc cataatgtac cccacttactggtatcacga ccctagaacc 720 ttccagctca gtgccgatga tatccaaagg atccagcatttgtatggaga aaaatgttca 780 tctgacatac cttaatgtta gcacagagga cttattcaacctgtctttca gggagtttat 840 tggaggatca aagaactgaa agcactagag cagccttggggactgctagg atgaagccct 900 aaagaatgca acctagtcag gttagctgaa ccgacactcaaaacgctact gagtcacaat 960 aaagattgtt ttaaagagta aaaaaaaaaa aaaaaaaaa 999<210> SEQ ID NO 8 <211> LENGTH: 261 <212> TYPE: PRT <213> ORGANISM: Homosapiens <300> PUBLICATION INFORMATION: <301> AUTHORS: Park, H.I., Ni,J., Gerkema, F.E., Liu, D., Belozero Sang, Q.X. <302> TITLE:Identification and characterization of human endometalloproteinase-26)from endometrial tumor <303> JOURNAL: J. Biol. Cehm. <304> VOLUME: 275<305> ISSUE: 27 <306> PAGES: 20540-205444 <307> DATE: 2000-03-23 <308>DATABASE ACCESSION NUMBER: GenBankAF248626 <309> DATABASE ENTRY DATE:2001-03-23 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: GenbankAF248646 <309> DATABASE ENTRY DATE: 2000-03-23 <400>SEQUENCE: 8 Met Gln Leu Val Ile Leu Arg Val Thr Ile Phe Leu Pro Trp CysPhe 1 5 10 15 Ala Val Pro Val Pro Pro Ala Ala Asp His Lys Gly Trp AspPhe Val 20 25 30 Glu Gly Tyr Phe His Gln Phe Phe Leu Thr Glu Lys Glu SerPro Leu 35 40 45 Leu Thr Gln Glu Thr Gln Thr Gln Leu Leu Gln Gln Phe HisArg Asn 50 55 60 Gly Thr Asp Leu Leu Asp Met Gln Met His Ala Leu Leu HisGln Pro 65 70 75 80 His Cys Gly Val Pro Asp Gly Ser Asp Thr Ser Ile SerPro Gly Arg 85 90 95 Cys Lys Trp Asn Lys His Thr Leu Thr Tyr Arg Ile IleAsn Tyr Pro 100 105 110 His Asp Met Lys Pro Ser Ala Val Lys Asp Ser IleTyr Asn Ala Val 115 120 125 Ser Ile Trp Ser Asn Val Thr Pro Leu Ile PheGln Gln Val Gln Asn 130 135 140 Gly Asp Ala Asp Ile Lys Val Ser Phe TrpGln Trp Ala His Glu Asp 145 150 155 160 Gly Trp Pro Phe Asp Gly Pro GlyGly Ile Leu Gly His Ala Phe Leu 165 170 175 Pro Asn Ser Gly Asn Pro GlyVal Val His Phe Asp Lys Asn Glu His 180 185 190 Trp Ser Ala Ser Asp ThrGly Tyr Asn Leu Phe Leu Val Ala Thr His 195 200 205 Glu Ile Gly His SerLeu Gly Leu Gln His Ser Gly Asn Gln Ser Ser 210 215 220 Ile Met Tyr ProThr Tyr Trp Tyr His Asp Pro Arg Thr Phe Gln Leu 225 230 235 240 Ser AlaAsp Asp Ile Gln Arg Ile Gln His Leu Tyr Gly Glu Lys Cys 245 250 255 SerSer Asp Ile Pro 260

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence that encodes a polypeptide comprising an amino acidsequence homologous to sequences selected from the group consisting of:SEQ ID NO:4 to SEQ ID NO:6; said nucleic acid molecule encoding at leasta portion of MMP, with the proviso that the nucleotide sequence is notSEQ ID NO:7.
 2. The isolated nucleic acid molecule of claim 1 comprisinga sequence that encodes a polypeptide comprising a sequence selectedfrom the group consisting of SEQ ID NO:4 to SEQ ID NO:6.
 3. The isolatednucleic acid molecule of claim 1 comprising a sequence homologous to asequence selected from the group consisting of SEQ ID NO:1 to SEQ IDNO:3.
 4. The isolated nucleic acid molecule of claim 1 comprising asequence selected from the group of sequences consisting of SEQ ID NO:1to SEQ ID NO:3.
 5. The isolated nucleic acid molecule of claim 1 whereinsaid nucleic acid molecule is DNA.
 6. The isolated nucleic acid moleculeof claim 1 wherein said nucleic acid molecule is RNA.
 7. An expressionvector comprising a nucleic acid molecule of any one of claims 1 to 4.8. The expression vector of claim 7 wherein said nucleic acid moleculecomprises a sequence selected from the group of sequences consisting ofSEQ ID NO:1 to SEQ ID NO:3.
 9. The expression vector of claim 7 whereinsaid vector is a plasmid.
 10. The expression vector of claim 7 whereinsaid vector is a viral particle.
 11. The expression vector of claim 10wherein said vector is selected from the group consisting ofadenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses,adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, andretroviruses.
 12. The expression vector of claim 7 wherein said nucleicacid molecule is operably connected to a promoter selected from thegroup consisting of simian virus 40, mouse mammary tumor virus, longterminal repeat of human immunodeficiency virus, maloney virus,cytomegalovirus immediate early promoter, Epstein Barr virus, roussarcoma virus, human actin, human myosin, human hemoglobin, human musclecreatine, and human metalothionein.
 13. A host cell transformed with anexpression vector of claim
 7. 14. The transformed host cell of claim 13wherein said cell is a bacterial cell.
 15. The transformed host cell ofclaim 14 wherein said bacterial cell is E. coli.
 16. The transformedhost cell of claim 13 wherein said cell is yeast.
 17. The transformedhost cell of claim 16 wherein said yeast is S. cerevisiae.
 18. Thetransformed host cell of claim 13 wherein said cell is an insect cell.19. The transformed host cell of claim 18 wherein said insect cell is S.frugiperda.
 20. The transformed host cell of claim 13 wherein said cellis a mammalian cell.
 21. The transformed host cell of claim 20 whereinmammalian cell is selected from the group consisting of chinese hamsterovary cells, HeLa cells, African green monkey kidney cells, humanHEK-293 cells, and murine 3T3 fibroblasts.
 22. An isolated nucleic acidmolecule comprising at least 10 nucleotides, said nucleic acid moleculecomprising a nucleotide sequence complementary to at least a portion ofa sequence selected from the group of sequences consisting of SEQ IDNO:1 to SEQ ID NO:3.
 23. The nucleic acid molecule of claim 22 whereinsaid molecule is an antisense oligonucleotide directed to a region of asequence selected from the group of sequences consisting of SEQ ID NO:1to SEQ ID NO:3.
 24. The nucleic acid molecule of claim 23 wherein saidoligonucleotide is directed to a regulatory region of a sequenceselected from the group of sequences consisting of SEQ ID NO:1 to SEQ IDNO:3.
 25. A composition comprising a nucleic acid molecule of any one ofclaims 1 to 4 or 22 and an acceptable carrier or diluent.
 26. Acomposition comprising a recombinant expression vector of claim 7 and anacceptable carrier or diluent.
 27. A method of producing a polypeptidethat comprises a sequence selected from the group of sequencesconsisting SEQ ID NO:4 to SEQ ID NO:6, and homologs thereof, said methodcomprising the steps of: a) introducing a recombinant expression vectorof claim 8 into a compatible host cell; b) growing said host cell underconditions for expression of said polypeptide; and c) recovering saidpolypeptide.
 28. The method of claim 27 wherein said host cell is lysedand said polypeptide is recovered from the lysate of said host cell. 29.The method of claim 27 wherein said polypeptide is recovered bypurifying the culture medium without lysing said host cell.
 30. Anisolated polypeptide encoded by a nucleic acid molecule of claim 1 withthe proviso that the polypeptide does not comprise the amino acidsequence of SEQ ID NO:8.
 31. The polypeptide of claim 30 wherein saidpolypeptide comprises a sequence selected from the group of sequencesconsisting of SEQ ID NO:4 to SEQ ID NO:6.
 32. The polypeptide of claim30 wherein said polypeptide comprises an amino acid sequence homologousto a sequence selected from the group of sequences consisting of SEQ IDNO:4 to SEQ ID NO:6, with the proviso that the polypeptide does notcomprise an amino acid sequence of SEQ ID NO:8.
 33. The polypeptide ofclaim 32 wherein said sequence homologous to a sequence selected fromthe group of sequences consisting of SEQ ID NO:4 to SEQ ID NO:6comprises at least one conservative amino acid substitution compared tothe sequences in the group of sequences consisting of SEQ ID NO:4 to SEQID NO:6.
 34. The polypeptide of claim 30 wherein said polypeptidecomprises an allelic variant of a polypeptide with a sequence selectedfrom the group of sequences consisting of SEQ ID NO:4 to SEQ ID NO:6.35. A composition comprising a polypeptide of claim 34 and an acceptablecarrier or diluent.
 36. An isolated antibody which binds to an epitopeon a polypeptide of claim
 30. 37. The antibody of claim 36 wherein saidantibody is a monoclonal antibody.
 38. A composition comprising anantibody of claim 36 and an acceptable carrier or diluent.
 39. A methodof inducing an immune response in a mammal against a polypeptide ofclaim 30 comprising administering to said mammal an amount of saidpolypeptide sufficient to induce said immune response.
 40. A method foridentifying a compound which binds MMP comprising the steps of: a)contacting MMP with a compound; and b) determining whether said compoundbinds MMP.
 41. The method of claim 40 wherein the MMP comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:4 to SEQID NO:6.
 42. The method of claim 40 wherein binding of said compound toMMP is determined by a protein binding assay.
 43. The method of claim 40wherein said protein binding assay is selected from the group consistingof a gel-shift assay, Western blot, radiolabeled competition assay,phage-based expression cloning, co-fractionation by chromatography,co-precipitation, cross linking, interaction trap/two-hybrid analysis,southwestern analysis, and ELISA.
 44. A compound identified by themethod of claim
 40. 45. A method for identifying a compound which bindsa nucleic acid molecule encoding MMP comprising the steps of: a)contacting said nucleic acid molecule encoding MMP with a compound; andb) determining whether said compound binds said nucleic acid molecule.46. The method of claim 45 wherein binding is determined by a gel-shiftassay.
 47. A compound identified by the method of claim
 45. 48. A methodfor identifying a compound which modulates the activity of MMPcomprising the steps of: a) contacting MMP with a compound; and b)determining whether MMP activity has been modulated.
 49. The method ofclaim 48 wherein the MMP comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:4 to SEQ ID NO:6.
 50. The method ofclaim 48 wherein said activity is proteinase activity.
 51. The method ofclaim 48 wherein said activity is collagenase activity.
 52. A compoundidentified by the method of claim
 48. 53. A method of identifying ananimal homolog of MMP comprising the steps: a) comparing the nucleicacid sequences of the animal with a sequence selected from the group ofsequence consisting of SEQ ID NO:1 to SEQ ID NO:3, and portions thereof,said portions being at least 10 nucleotides; and b) identifying nucleicacid sequences of the animal that are homologous to said sequenceselected from the group sequence consisting of SEQ ID NO:1 to SEQ IDNO:3, and portions thereof, said portions comprising at least 10nucleotides.
 54. The method of claim 53 wherein comparing the nucleicacid sequences of the animal with a sequence selected from the group ofsequences consisting of SEQ ID NO:1 to SEQ ID NO:3, and portionsthereof, said portions being at least 10 nucleotides, is performed byDNA hybridization.
 55. The method of claim 53 wherein comparing thenucleic acid sequences of the animal with a sequence selected from thegroup of sequences consisting of SEQ ID NO:1 to SEQ ID NO:3, andportions thereof, said portions being at least 10 nucleotides, isperformed by computer homology search.
 56. A method of screening a humansubject to diagnose a disorder affecting the brain or geneticpredisposition therefor, comprising the steps of: (a) assaying nucleicacid of a human subject to determine a presence or an absence of amutation altering an amino acid sequence, expression, or biologicalactivity of at least one MMP that is expressed in the brain, wherein theMMP comprises an amino acid sequence selected from the group consistingof SEQ ID NO:4 to SEQ ID NO:6, and allelic variants thereof, and whereinthe nucleic acid corresponds to a gene encoding the MMP; and (b)diagnosing the disorder or predisposition from the presence or absenceof said mutation, wherein the presence of a mutation altering the aminoacid sequence, expression, or biological activity of the MMP in thenucleic acid correlates with an increased risk of developing thedisorder.
 57. A method according to claim 56, wherein the disease is amental disorder.
 58. A method according to claim 56, wherein theassaying step comprises at least one procedure selected from the groupconsisting of: a) comparing nucleotide sequences from the human subjectand reference sequences and determining a difference of at least anucleotide of at least one codon between the nucleotide sequences fromthe human subject that encodes a MMP reference sequence; (b) performinga hybridization assay to determine whether nucleic acid from the humansubject has a nucleotide sequence identical to or different from one ormore reference sequences; (c) performing a polynucleotide migrationassay to determine whether nucleic acid from the human subject has anucleotide sequence identical to or different from one or more referencesequences; and (d) performing a restriction endonuclease digestion todetermine whether nucleic acid from the human subject has a nucleotidesequence identical to or different from one or more reference sequences.59. A method according to claim 58 wherein the assaying step comprises:performing a polymerase chain reaction assay to amplify nucleic acidcomprising MMP coding sequence, and determining nucleotide sequence ofthe amplified nucleic acid.
 60. A method of screening for an MMPhereditary mental disorder genotype in a human patient, comprising thesteps of: (a) providing a biological sample comprising nucleic acid fromsaid patient, said nucleic acid including sequences corresponding toalleles of MMP; and (b) detecting the presence of one or more mutationsin the MMP allele; wherein the presence of a mutation in a MMP allele isindicative of a hereditary mental disorder genotype.
 61. The methodaccording to claim 60 wherein said biological sample is a cell sample.62. The method according to claim 60 wherein said detecting the presenceof a mutation comprises sequencing at least a portion of said nucleicacid, said portion comprising at least one codon of said MMP allele,said portion comprising at least 10 nucleotides.
 63. The methodaccording to claim 60 wherein said nucleic acid is DNA.
 64. The methodaccording to claim 60 wherein said nucleic acid is RNA.
 65. A kit forscreening a human subject to diagnose a mental disorder or a geneticpredisposition therefor, comprising, in association: (a) anoligonucleotide useful as a probe for identifying polymorphisms in ahuman MMP gene, the oligonucleotide comprising 6-50 nucleotides in asequence that is identical or complementary to a sequence of a wild typehuman MMP gene sequence or MMP coding sequence, except for one sequencedifference selected from the group consisting of a nucleotide addition,a nucleotide deletion, or nucleotide substitution; and (b) a mediapackaged with the oligonucleotide, said media containing information foridentifying polymorphisms that correlate with mental disorder or agenetic predisposition therefor, the polymorphisms being identifiableusing the oligonucleotide as a probe.
 66. A method of identifying a MMPallelic variant that correlates with a mental disorder, comprising thesteps of: (a) providing a biological sample comprising nucleic acid froma human patient diagnosed with a mental disorder, or from the patient'sgenetic progenitors or progeny; (b) detecting in the nucleic acid thepresence of one or more mutations in an MMP that is expressed in thebrain, wherein the MMP comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:4 to SEQ ID NO:6, and allelic variantsthereof, and wherein the nucleic acid includes sequence corresponding tothe gene or genes encoding MMP; wherein the one or more mutationsdetected indicates an allelic variant that correlates with a mentaldisorder.
 67. A purified and isolated polynucleotide comprising anucleotide sequence encoding a MMP allelic variant identified accordingto claim
 66. 68. A host cell transformed or transfected with apolynucleotide according to claim 67 or with a vector comprising thepolynucleotide.
 69. A purified polynucleotide comprising a nucleotidesequence encoding MMP of a human with a mental disorder; wherein saidpolynucleotide hybridizes to the complement of a sequence selected fromthe group consisting of SEQ ID NO:1 to SEQ ID NO:3 under the followinghybridization conditions: (a) hybridization for 16 hours at 42° C. in ahybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10%dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in awash solution comprising 0.1×SSC and 1% SDS; and wherein thepolynucleotide encodes an MMP amino acid sequence of the human thatdiffers from the sequence selected from the group consisting of SEQ IDNO:4 to SEQ ID NO:6 by at least one residue, with the proviso that theamino acid sequence is not SEQ ID NO:8.
 70. A vector comprising apolynucleotide according to claim
 69. 71. A host cell that has beentransformed or transfected with a polynucleotide according to claim 69and that expresses the MMP protein encoded by the polynucleotide.
 72. Ahost cell according to claim 71 that has been co-transfected with apolynucleotide encoding the MMP amino acid sequence set forth in asequence selected from the group consisting of SEQ ID NO:1 to SEQ IDNO:3 and that expresses the MMP protein having the amino acid sequenceset forth in SEQ ID NO:4 to SEQ ID NO:6.
 73. A method for identifying amodulator of biological activity of MMP comprising the steps of: a)contacting a cell according to claim 72 in the presence and in theabsence of a putative modulator compound; b) measuring MMP biologicalactivity in the cell; wherein decreased or increased MMP biologicalactivity in the presence versus absence of the putative modulator isindicative of a modulator of biological activity.
 74. A method toidentify compounds useful for the treatment of a mental disorder, saidmethod comprising the steps of: (a) contacting a composition comprisingMNP with a compound suspected of binding MMP; (b) detecting bindingbetween MMP and the compound suspected of binding MMP; wherein compoundsidentified as binding MMP are candidate compounds useful for thetreatment of a mental disorder.
 75. A method for identifying a compounduseful as a modulator of binding between MMP and a binding partner ofMMP comprising the steps of: (a) contacting the binding partner and acomposition comprising MMP in the presence and in the absence of aputative modulator compound; (b) detecting binding between the bindingpartner and MMP; wherein decreased or increased binding between thebinding partner and MMP in the presence of the putative modulator, ascompared to binding in the absence of the putative modulator isindicative a modulator compound useful for the treatment of a mentaldisorder.
 76. A method according to claim 74 or 75 wherein thecomposition comprises a cell expressing MMP on its surface.
 77. A methodaccording to claim 76 wherein the composition comprises a celltransformed or transfected with a polynucleotide that encodes MMP.